α-amylase and α-amylase variants

ABSTRACT

The invention relates to a novel Termamyl-like α-amylase, and Termamyl-like α-amylases comprising mutations in two, three, four, five or six regions/positions. The variants have increased thermostability at acidic pH and/or at low Ca 2+  concentrations (relative to the parent). The invention also relates to a DNA construct comprising a DNA sequence encoding an α-amylase variant of the invention, a recombinant expression vector which carries a DNA construct of the invention, a cell which is transformed with a DNA construct of the invention, the use of an α-amylase variant of the invention for washing and/or dishwashing, textile desizing, starch liquefaction, a detergent additive comprising an α-amylase variant of the invention, a manual or automatic dishwashing detergent composition comprising an α-amylase variant of the invention, a method for generating a variant of a parent Termamyl-like α-amylase, which variant exhibits increased thermostability at acidic pH and/or at low Ca 2+  concentrations (relative to the parent).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. application Ser. No.09/170,670 filed on Oct. 13, 1998 now U.S. Pat. No. 6,187,576 issued onFeb. 13, 2001 which claims priority of Provisional application No.60/063,306 filed Oct. 28, 1997 and Danish application no. 1172/97 filedon Oct. 13, 1997, and further claims priority of Danish application no.PA 1999 00439 filed Mar. 31, 1999, the contents of which are fullyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates a novel α-amylase within the family ofTermamyl-like α-amylases suitable for detergents. The invention alsorelates to variants (mutants) of parent Termamyl-like α-amylases,notably variants exhibiting increased thermostability at acidic pHand/or at low Ca²⁺ concentrations (relative to the parent) which areadvantageous with respect to applications of the variants in, industrialstarch processing particularly (e.g., starch liquefaction orsaccharification). Said α-amylase and α-amylase variants of theinvention may advantageously also be used in detergents.

BACKGROUND OF THE INVENTION

α-Amylases (α-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute agroup of enzymes which catalyze hydrolysis of starch and other linearand branched 1,4-glucosidic oligo- and polysaccharides.

There is a very extensive body of patent and scientific literaturerelating to this industrially very important class of enzymes. A numberof α-amylase such as Termamyl-like α-amylases variants are known from,e.g., WO 90/11352, WO 95/10603, WO 95/26397, WO 96/23873 and WO96/23874.

Among more recent disclosures relating to α-amylases, WO 96/23874provides three-dimensional, X-ray crystal structural data for aTermamyl-like α-amylase which consists of the 300 N-terminal amino acidresidues of the B. amyloliquefaciens α-amylase and amino acids 301-483of the C-terminal end of the B. licheniformis α-amylase comprising theamino acid sequence (the latter being available commercially under thetradename Termamyl™), and which is thus closely related to theindustrially important Bacillus α-amylases (which in the present contextare embraced within the meaning of the term “Termamyl-like α-amylases”,and which include, inter alia, the B. licheniformis, B.amyloliquefaciens and B. stearothermophilus α-amylases). WO 96/23874further describes methodology for designing, on the basis of an analysisof the structure of a parent Termamyl-like α-amylase, variants of theparent Termamyl-like α-amylase which exhibit altered properties relativeto the parent.

WO 95/35382 (Gist Brocades B.V.) concerns amylolytic enzymes derivedfrom B. licheniformis with improved properties allowing reduction of theCa²⁺ concentration under application without a loss of performance ofthe enzyme. The amylolytic enzyme comprises one or more amino acidchanges at positions selected from the group of 104, 128, 187, 188 ofthe B. licheniformis α-amylase sequence.

WO 96/23873 (Novo Nordisk) discloses Termamyl-like α-amylase variantswhich have increased thermostability obtained by pairwise deletion inthe region R181*, G182*, T183* and G184* of the sequence shown in SEQ IDNO: 1 herein.

WO 97/00324 (KAO) disclose a gene encoding an alkaline liquefyingα-amylase derived from Bacillus sp. strain KSM-AP1378 with the depositedno. FERM BP-3048 suitable for detergents.

BRIEF DISCLOSURE OF THE INVENTION

The present invention relates to a novel α-amylase and to novelα-amylolytic variants (mutants) of a Termamyl-like α-amylase, inparticular variants exhibiting increased thermostability (relative tothe parent) which are advantageous in connection with the industrialprocessing of starch (starch liquefaction, saccharification and thelike). The novel α-amylase is suitable for laundry washing and dishwashas is has a high activity under wash conditions at alkaline pHs in therange 9-11.

The inventors have surprisingly found out that in case of combining two,three, four, five or six mutations (will be described below), thethermostability of Termamyl-like α-amylases is increased at acidic pHand/or at low Ca²⁺ concentration in comparison to single mutations, suchas the mutation disclosed in WO 96/23873 (Novo Nordisk), i.e., pairwisedeletion in the region R181*, G182*, T183* and G184* of the sequenceshown in SEQ ID NO: 1 herein.

The invention further relates to DNA constructs encoding variants of theinvention, to composition comprising variants of the invention, tomethods for preparing variants of the invention, and to the use ofvariants and compositions of the invention, alone or in combination withother α-amylolytic enzymes, in various industrial processes, e.g.,starch liquefaction.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an alignment of the amino acid sequences of nine parentTermamyl-like α-amylases. The numbers on the Extreme left designate therespective amino acid sequences as follows:

1: SEQ ID NO: 2,

2: Bacillus sp. strain KSM-AP1378 disclosed in WO 97/00324

3: SEQ ID NO: 1,

4: SEQ ID NO: 5,

5: SEQ ID NO: 4,

6: SEQ ID NO: 3.

7: Partial α-amylase sequence

8: SEQ ID NO: 24

9: SEQ ID NO: 26.

FIG. 2 shows the pH Profile of the AA560 a-amylase compared to the SP722and SP690 α-amylases. The pH profile was measured at 37° C. The activityis shown in absolute values as Abs650/mg.

FIG. 3 shows the Temperature Profile of the AA560 α-amylase compared tothe SP722 and SP690 α-amylases. The temperature profile shown asAbs650/mg.

FIG. 4 shows the wash performance of AA560 in the AP Model Detergent 97in comparison to SP722, SP690 and Termamyl®.

FIG. 5 shows the wash performance of AA560 in the Omo Multi Acao incomparison to SP722, SP690 and Termamyl®.

FIG. 6 shows the wash performance of AA560 in the Omo Concentrated incomparison to SP722, SP690 and Termamyl®.

FIG. 7 shows the wash performance of AA560 in the Ariel Futur liquid incomparison to SP722, SP690 and Termamyl®.

DETAILED DISCLOSURE OF THE INVENTION α-Amylase Activity Determination

α-Amylases (α-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute agroup of enzymes which catalyze hydrolysis of starch and other linearand branched 1,4-glucosidic oligo- and polysaccharides. For purposes ofthe present invention, α-amylase activity may be determined using thePhadebas assay, the pNPG7 assay and the BS-α-amylase activity assaydescribed below in the “Materials and Methods” section.

The Novel α-amylase

Microbial Source

The novel alkaline α-amylase of the invention may be derived from astrain of Bacillus. Preferred strains are of Bacillus sp. DSM 12649 (theAA560 α-amylase) or Bacillus sp. DSM 12648 (the AA349 α-amylase). Thesestrains were deposited on Jan. 25^(th), 1999 by the inventors under theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure atDeutshe Sammmlung von Microorganismen und Zellkulturen GmbH (DSMZ),Mascheroder Weg 1b, D-38124 Braunschweig DE.

Escherichia coli strains termed NN049467 and NN049470 containing theα-amylase genes in plasmids pLiH1274 (AA349) and plasmid pTVB299 (AA560)have also been deposited on Apr. 7^(th), 1999 under the terms of theBudapest Treaty with the Deutshe Sammmlung von Microorganismen undZellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig DE,and given the accession numbers DSM12761 and DSM12764, respectively.

Homology of Enzyme

In a first embodiment, the present invention relates to isolatedpolypeptides having an amino acid sequence which has a degree ofidentity to amino acids 1 to 485 of SEQ ID NO: 24 or SEQ ID NO: 26(i.e., the mature polypeptide) of at least about 96%, preferably atleast about 97%, more preferably at least about 98%, even morepreferably at least about 99%, which have α-amylase activity(hereinafter “homologous polypeptides”). In a preferred embodiment, thehomologous polypeptides have an amino acid sequence which differs byfive amino acids, preferably by four amino acids, more preferably bythree amino acids, even more preferably by two amino acids, and mostpreferably by one amino acid from amino acids 1 to 485 of SEQ ID NO: 24or SEQ ID NO: 26. It is to be noted that SEQ ID NO: 24 and SEQ ID NO: 26are identical. However, the DNA sequences, i.e., SEQ ID NO: 23 and SEQID NO: 25, respectively, encoding the α-amylase of the invention shownin SEQ ID NO: 24 and SEQ ID NO: 26 are not identical.

The amino acid sequence homology may be determined as the degree ofidentity between the two sequences indicating a derivation of the firstsequence from the second. The homology may suitably be determined bymeans of computer programs known in the art. Thus, GAP provided in GCGversion 8 (Needleman, S. B. and Wunsch, C. D., (1970), Journal ofMolecular Biology, 48, 443-453) may be used for a pairwise alignment ofthe sequences and calculation of the degree of identity or degree ofhomology using the default settings. Alternatively, Gap from GCG version9 may be used with a translated version 8 peptide scoring matrix, a gapcreation penalty of 30, a gap extension penalty of 1 using ntol's matrix(http://plasmid/˜bioweb/matrix/) without end gap penalty.

Homology (identity) of the Novel α-amylase to Known Bacillus sp.α-amylases

A homology search of known sequences showed homologies for the sequencesof the invention with a number of Bacillus amylases in the range 65-95%on amino acid basis determined as described above.

Specifically, the most homologous α-amylases found are SP690 (SEQ ID NO:1 of U.S. Pat. No. 5,856,164 which is about 87% homologous), SP722 (SEQID NO: 2 of U.S. Pat. No. 5,856,164 which is about 87% homologous), themature part (i.e., amino acids no. 31-516) of the α-amylase obtainedfrom Bacillus sp. KSM-AP1378 disclosed as SEQ ID NO: 2 of WO 97/00324which is about 86% homologous, and the α-amylase disclosed in Tsukamotoet. al., (1988), Biochem. Biophys. Res Commun. 151, p. 25-33) which isabout 95% homologous to SEQ ID NO: 24 and SEQ ID NO: 26 determined asdescribe above.

Preferably, the polypeptides of the present invention comprise the aminoacid sequence of SEQ ID NO: 24 or SEQ ID NO: 26 or allelic variantsthereof; or fragments thereof that has α-amylase activity. SEQ ID NO: 24and SEQ ID NO: 26 show the mature part of the alkaline α-amylase of theinvention.

A fragment of SEQ ID NO: 24 or SEQ ID NO: 26 are polypeptides having oneor more amino acids deleted from the amino and/or carboxyl terminus ofthis amino acid sequence.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

The amino acid sequences of the homologous polypeptides may differ fromthe amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26 by aninsertion or deletion of one or more amino acid residues and/or thesubstitution of one or more amino acid residues by different amino acidresidues. Preferably, amino acid changes are of a minor nature, that isconservative amino acid substitutions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof one to about 30 amino acids; small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue; a small linkerpeptide of up to about 20-25 residues; or a small extension thatfacilitates purification by changing net charge or another function,such as a poly-histidine tract, an antigenic epitope or a bindingdomain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these inreverse.

In a second embodiment, the present invention relates to isolatedpolypeptides having α-amylase activity which are encoded by nucleic acidsequences which hybridize under medium stringency conditions, preferablymedium-high stringency conditions, more preferably high stringencyconditions, and most preferably very high stringency conditions with anucleic acid probe which hybridizes under the same conditions with (i)the nucleic acid sequence of SEQ ID NO: 23 or SEQ ID NO: 25, (ii) thecDNA sequence of SEQ ID NO: 23 or SEQ ID NO: 25, (iii) a subsequence of(i) or (ii), or (iv) a complementary strand of (i), (ii), or (iii) (J.Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). Thesubsequence of SEQ ID NO: 23 or SEQ ID NO: 25 may be at least 100nucleotides or preferably at least 200 nucleotides. Moreover, thesubsequence may encode a polypeptide fragment which has α-amylaseactivity. The polypeptides may also be allelic variants or fragments ofthe polypeptides that have α-amylase activity.

The nucleic acid sequence of SEQ ID NO: 23 or SEQ ID NO: 25 or asubsequence thereof, as well as the amino acid sequence of SEQ ID NO: 24or SEQ ID NO: 26 or a fragment thereof, may be used to design a nucleicacid probe to identify and clone DNA encoding polypeptides havingα-amylase activity from strains of different genera or species accordingto methods well known in the art. In particular, such probes can be usedfor hybridization with the genomic or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, preferably at least 25, and more preferably at least 35 nucleotidesin length. Longer probes can also be used. Both DNA and RNA probes canbe used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²p, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA which hybridizes with the probes described aboveand which encodes a polypeptide having α-amylase activity. Genomic orother DNA from such other organisms may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA which is homologous with SEQ ID NO: 23or SEQ ID NO: 25 or subsequences thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleic acid sequence hybridizes to a nucleic acid probe correspondingto the nucleic acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 25,its complementary strand, or subsequences thereof, under medium to veryhigh stringency conditions. Molecules to which the nucleic acid probehybridizes under these conditions are detected using X-ray film.

In another preferred embodiment, the nucleic acid probe is the nucleicacid sequence contained in plasmids pLiH1274 (AA349) or pTVB299 (AA560)which are contained in Escherichia coli DSM12761 or Escherichia coliDSM12764, respectively, or, wherein the nucleic acid sequence encodes apolypeptide having acid α-amylase activity of the invention and shown inSEQ ID NO: 24 and SEQ ID NO: 26, respectively.

For long probes of at least 100 nucleotides in length, medium to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 55° C. (medium stringency), preferablyat least at 60° C. (medium-high stringency), more preferably at least at65° C. (high stringency), and most preferably at least at 70° C. (veryhigh stringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at 5° C. to 10° C. belowthe calculated T_(m) using the calculation according to Bolton andMcCarthy (1962, Proceedings of the National Academy of Sciences USA48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40,1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasicphosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standardSouthern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

In a third embodiment, the present invention relates to isolatedpolypeptides, i.e., the polypeptides shown in SEQ ID NO: 24 or SEQ IDNO: 26, having the following physicochemical properties:

A pH optimum (see FIG. 2) determined using the Phadebas method (37° C.)was found to be in the range between pH 8 and 9, more precisely at about8.5.

A temperature optimum (See FIG. 3) determined using the Phasebas method(pH 9.0) was found to be in the range between 55 and 65° C., moreprecisely about 60° C.

A pI between 7-8 (See Table 1 in Example 11) was determined byisoelectric focusing (Pharmacia, Ampholine, pH 3.5-9.3).

A specific activity (see Table 1 of Example 11) of 35,000 NU/ml wasdetermined using the Phadebas method and 6,000 NU/ml using the pNPG7method.

The α-amylase of the present invention have at least 20%, preferably atleast 40%, more preferably at least 60%, even more preferably at least80%, even more preferably at least 90%, and most preferably at least100% of the α-amylase activity of the mature α-amylase shown in SEQ IDNO: 24 and SEQ ID NO: 26.

An α-amylase of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the α-amylase encoded by the nucleic acid sequence isproduced by the source or by a cell in which the nucleic acid sequencefrom the source has been inserted.

An α-amylase of the present invention is a bacterial polypeptide. Forexample, the polypeptide may be a gram positive bacterial polypeptidesuch as a Bacillus polypeptide, e.g., a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacilluscoagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, orBacillus thuringiensis polypeptide; or a Streptomyces polypeptide, e.g.,a Streptomyces lividans or Streptomyces murinus polypeptide; or a gramnegative bacterial polypeptide, e.g., an E. coli or a Pseudomonas sp.polypeptide.

In another preferred embodiment, the polypeptide is a Bacillus sp.polypeptide, more preferred embodiment, the polypeptide is a Bacillussp. DSM 12648 or Bacillus sp. DSM 12649 polypeptide, e.g., thepolypeptides with the amino acid sequence of SEQ ID NO: 24 and SEQ IDNO: 26, respectively.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The nucleic acid sequence may then be derived by similarlyscreening a genomic or cDNA library of another microorganism. Once anucleic acid sequence encoding a polypeptide has been detected with theprobe(s), the sequence may be isolated or cloned by utilizing techniqueswhich are known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

As defined herein, an “isolated” polypeptide is a polypeptide which isessentially free of other non-α-amylase polypeptides, e.g., at leastabout 20% pure, preferably at least about 40% pure, more preferablyabout 60% pure, even more preferably about 80% pure, most preferablyabout 90% pure, and even most preferably about 95% pure, as determinedby SDS-PAGE.

Polypeptides encoded by nucleic acid sequences of the present inventionalso include fused polypeptides or cleavable fusion polypeptides inwhich another polypeptide is fused at the N-terminus or the C-terminusof the polypeptide or fragment thereof. A fused polypeptide is producedby fusing a nucleic acid sequence (or a portion thereof) encodinganother polypeptide to a nucleic acid sequence (or a portion thereof) ofthe present invention. Techniques for producing fusion polypeptides areknown in the art, and include ligating the coding sequences encoding thepolypeptides so that they are in frame and that expression of the fusedpolypeptide is under control of the same promoter(s) and terminator.

Mutants of the Novel α-amylase

Specifically contemplated mutants of the novel α-amylase shown in SEQ IDNO: 24 (or SEQ ID NO: 26) are described in the following. A mutantα-amylase of the invention is characterized by the fact that one or moreof the methionine amino acid residues is exchanged with any amino acidresidue except for Cys and Met. Thus, according to the invention theamino acid residues to replace the methionine amino acid residue are thefollowing: Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe,Pro, Ser, Thr, Trp, Tyr, and Val.

A preferred embodiment of the mutant α-amylase of the invention ischaracterized by the fact that one or more of the methionine amino acidresidues is (are) exchanged with a Leu, Thr, Ala, Gly, Ser, Ile, or Valamino acid residue, preferably a Leu, Thr, Ala, or Gly amino acidresidue. In this embodiment a very satisfactory activity level andstability in the presence of oxidizing agents is obtained. Specificallythis means that one or more of the methiones in the following positionmay be replaced or deleted using any suitable technique known in theart, including especially site directed mutagenesis and gene shuffling.Contemplated position, using the SEQ ID NO: 24 numbering, are: 9, 10,105, 116, 202, 208, 261, 309, 323, 382, 430, 440.

In a preferred embodiment of the mutant α-amylase of the invention ischaracterized by the fact that the methionine amino acid residue atposition 202 is exchanged with any of amino acid residue expect for Cysand Met, preferably with a Leu, Thr, Ala, Gly, Ser, Ile, or Asp.

Other contemplated preferred mutations include deletion of one, two ormore residues of amino acids R181, G182, D183 or G184, K185, G186 orsubstitution of one or more of these residues. A preferred mutation isthe deletion of D183-G184. Particularly relevant mutations aresubstitutions of G186 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile,Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. A particularlypreferred substitution is G186R.

Also contemplated is substitution of N195 with Ala, Arg, Asn, Asp, Cys,Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr,and Val. A particularly interesting substitution is N195F.

The following combinations of the above mentioned mutations include:deletion of D183−G184+N195F, deletion of D183−G184+G186R, deletion ofD183−G184+G186R+N195F and G186R+N195F.

Nucleic Acid Sequences

The present invention also relates to isolated nucleic acid sequenceswhich encode a polypeptide of the present invention. In a preferredembodiment, the nucleic acid sequence is set forth in SEQ ID NO: 23 orSEQ ID NO: 25. In another more preferred embodiment, the nucleic acidsequence is the sequence contained in plasmid pLiH1274 (AA349) orplasmid pTVB299 (AA560) that is contained in Escherichia coli DSM12761and Escherichia coli DSM12764, respectively. In another preferredembodiment, the nucleic acid sequence is the mature polypeptide codingregion of SEQ ID NO: 23 or SEQ ID NO: 25. The present invention alsoencompasses nucleic acid sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO: 24 which differ from SEQ ID NO: 23 orSEQ ID NO: 25 by virtue of the degeneracy of the genetic code. Thepresent invention also relates to subsequences of SEQ ID NO: 23 or SEQID NO: 25 which encode fragments of SEQ ID NO: 24 or SEQ ID NO: 26,respectively, that have α-amylase activity.

Subsequences of SEQ ID NO: 23 or SEQ ID NO: 25 are nucleic acidsequences encompassed by SEQ ID NO: 23 or SEQ ID NO: 25 except that oneor more nucleotides from the 5′ and/or 3′ end have been deleted.

The present invention also relates to mutant nucleic acid sequencescomprising at least one mutation in the mature polypeptide codingsequence of SEQ ID NO: 1 or SEQ ID NO: 3, in which the mutant nucleicacid sequence encodes a polypeptide which consists of amino acids 1 to485 of SEQ ID NO: 24 or SEQ ID NO: 26.

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide are known in the art and include isolation from genomicDNA, preparation from cDNA, or a combination thereof. The cloning of thenucleic acid sequences of the present invention from such genomic DNAcan be effected, e.g., by using the well known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used. The nucleic acidsequence may be cloned from a strain of Bacillus, or another or relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the nucleic acid sequence.

The term “isolated nucleic acid sequence” as used herein refers to anucleic acid sequence which is essentially free of other nucleic acidsequences, e.g., at least about 20% pure, preferably at least about 40%pure, more preferably at least about 60% pure, even more preferably atleast about 80% pure, and most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

Homology of DNA Sequence Encoding the Enzyme

The present invention also relates to nucleic acid sequences which havea degree of homology to the mature polypeptide coding sequence of SEQ IDNO: 23 (i.e., nucleotides 1 to 1458) or SEQ ID NO: 25 (i.e., nucleotide1 to 1458) of at least about 96% homology on DNA level, preferably about97%, preferably about 98%, more preferably about 99% homology, whichencode an active polypeptide.

The DNA sequence homology may be determined as the degree of identitybetween the two sequences indicating a derivation of the first sequencefrom the second. The homology may suitably be determined by means ofcomputer programs known in the art such as GAP provided in the GCGprogram package (described above). Thus, Gap GCGv8 may be used with thefollowing default parameters: GAP creation penalty of 5.0 and GAPextension penalty of 0.3, default scoring matrix. GAP uses the method ofNeedleman/Wunsch/Sellers to make alignments.

Modification of a nucleic acid sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., variantsthat differ in specific activity, thermostability, pH optimum, or thelike. The variant sequence may be constructed on the basis of thenucleic acid sequence presented as the polypeptide encoding part of SEQID NO: 23 or SEQ ID NO: 25, e.g., a subsequence thereof, and/or byintroduction of nucleotide substitutions which do not give rise toanother amino acid sequence of the polypeptide encoded by the nucleicacid sequence, but which correspond to the codon usage of the hostorganism intended for production of the enzyme, or by introduction ofnucleotide substitutions which may give rise to a different amino acidsequence. For a general description of nucleotide substitution, see,e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by the isolated nucleic acidsequence of the invention, and therefore preferably not subject tosubstitution, may be identified according to procedures known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In thelatter technique, mutations are introduced at every positively chargedresidue in the molecule, and the resultant mutant molecules are testedfor [enzyme] activity to identify amino acid residues that are criticalto the activity of the molecule. Sites of substrate-enzyme interactioncan also be determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labelling (see, e.g., de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, Journal of MolecularBiology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The present invention also relates to isolated nucleic acid sequencesencoding a polypeptide of the present invention, which hybridize undermedium stringency conditions, preferably medium-high stringencyconditions, more preferably high stringency conditions, and mostpreferably very high stringency conditions with a nucleic acid probewhich hybridizes under the same conditions with the nucleic acidsequence of SEQ ID NO: 1 or SEQ ID NO: 3 or its complementary strand; orallelic variants and subsequences thereof (Sambrook et al., 1989,supra), as defined herein.

The present invention also relates to isolated nucleic acid sequencesproduced by (a) hybridizing a DNA under medium, medium-high, high, orvery high stringency conditions with the sequence of SEQ ID NO: 23 orSEQ ID NO: 25, or their complementary strands, or a subsequence thereof;and (b) isolating the nucleic acid sequence. The subsequence ispreferably a sequence of at least 100 nucleotides such as a sequencewhich encodes a polypeptide fragment which has α-amylase activity.

Methods for Producing Mutant Nucleic Acid Sequences

The present invention further relates to methods for producing a mutantnucleic acid sequence, comprising introducing at least one mutation intothe mature polypeptide coding sequence of SEQ ID NO: 23 or SEQ ID NO: 25or a subsequence thereof, wherein the mutant nucleic acid sequenceencodes a polypeptide which consists of 1 to 485 of SEQ ID NO: 24 or SEQID NO: 26 or a fragment thereof which has α-amylase activity.

The introduction of a mutation into the nucleic acid sequence toexchange one nucleotide for another nucleotide may be accomplished bysite-directed mutagenesis using any of the methods known in the art.Particularly useful is the procedure which utilizes a supercoiled,double stranded DNA vector with an insert of interest and two syntheticprimers containing the desired mutation. The oligonucleotide primers,each complementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with DpnI which isspecific for methylated and hemimethylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art may also be used. These otherprocedures include gene shuffling, e.g., as described in WO 95/22625(from Affymax Technologies N.V.) and WO 96/00343 (from Novo NordiskA/S).

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga nucleic acid sequence of the present invention operably linked to oneor more control sequences which direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences. Expression will be understood to include any stepinvolved in the production of the polypeptide including, but not limitedto, transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid which are combined and juxtaposed in a manner which would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term expression cassette when the nucleic acid constructcontains all the control sequences required for expression of a codingsequence of the present invention. The term “coding sequence” is definedherein as a portion of a nucleic acid sequence which directly specifiesthe amino acid sequence of its protein product. The boundaries of thecoding sequence are generally determined by a ribosome binding site(prokaryotes) or by the ATG start codon (eukaryotes) located justupstream of the open reading frame at the 5′ end of the mRNA and atranscription terminator sequence located just downstream of the openreading frame at the 3′ end of the mRNA. A coding sequence can include,but is not limited to, DNA, cDNA, and recombinant nucleic acidsequences.

An isolated nucleic acid sequence encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the nucleic acid sequenceprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifying nucleicacid sequences utilizing recombinant DNA methods are well known in theart.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for the expression of a polypeptideof the present invention. Each control sequence may be native or foreignto the nucleic acid sequence encoding the polypeptide. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide. The term “operably linked” is defined herein asa configuration in which a control sequence is appropriately placed at aposition relative to the coding sequence of the DNA sequence such thatthe control sequence directs the expression of a polypeptide.

Promoter Sequence

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences which mediate the expression of the polypeptide. Thepromoter may be any nucleic acid sequence which shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Terminator Sequence

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Signal Peptide

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Regulatory System

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a nucleic acid sequence of the present invention, a promoter,and transcriptional and translational stop signals. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the polypeptide at such sites.Alternatively, the nucleic acid sequence of the present invention may beexpressed by inserting the nucleic acid sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleic acid sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers whichconfer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol or tetracycline resistance. Suitable markers for yeasthost cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Aselectable marker for use in a filamentous fungal host cell may beselected from the group including, but not limited to, amds(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents thereof. Preferredfor use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptorayces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell genomeor autonomous replication of the vector in the cell independent of thegenome of the cell.

For integration into the host cell genome, the vector may rely on thenucleic acid sequence encoding the polypeptide or any other element ofthe vector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1permitting replication in Bacillus. Examples of origins of replicationfor use in a yeast host cell are the 2 micron origin of replication,ARS1, ARS4, the combination of ARS1 and CEN3, and the combination ofARS4 and CEN6. The origin of replication may be one having a mutationwhich makes its functioning temperature-sensitive in the host cell (see,e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA75: 1433).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga nucleic acid sequence of the invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising anucleic acid sequence of the present invention is introduced into a hostcell so that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular cells are bacterial cells such as gram positivebacteria including, but not limited to., a Bacillus cell, e.g., Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus clausii, Bacillus coagulans, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or aStreptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus,or gram negative bacteria such as E. coli and Pseudomonas sp. In apreferred embodiment, the bacterial host cell is a Bacillus lentus,Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtiliscell. In another preferred embodiment, the Bacillus cell is analkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

Methods of Production

The present invention also relates to methods for producing an α-amylaseof the present invention comprising (a) cultivating a strain, which inits wild-type form is capable of producing the polypeptide, to produce asupernatant comprising the polypeptide; and (b) recovering thepolypeptide. Preferably, the strain is of the genus Bacillus sp.

The present invention also relates to methods for producing an α-amylaseof the present invention comprising (a) cultivating a host cell underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

The present invention also relates to methods for producing an α-amylaseof the present invention comprising (a) cultivating a host cell underconditions conducive for production of the polypeptide, wherein the hostcell comprises a mutant nucleic acid sequence having at least onemutation in the mature polypeptide coding region of SEQ ID NO: 23 or SEQID NO: 25, wherein the mutant nucleic acid sequence encodes apolypeptide which consists of amino acids 1 to 485 of SEQ ID NO: 24 orSEQ ID NO: 26, and (b) recovering the polypeptide.

MUTANT α-AMYLASE

The present invention also relates to α-amylase mutants.

The Termamyl-like α-amylase

It is well known that a number of α-amylases produced by Bacillus spp.are highly homologous on the amino acid level. For instance, the B.licheniformis α-amylase comprising the amino acid sequence shown in SEQID NO: 4 (commercially available as Termamyl™) has been found to beabout 89% homologous with the B. amyloliquefaciens α-amylase comprisingthe amino acid sequence shown in SEQ ID NO: 5 and about 79% homologouswith the B. stearothermophilus α-amylase comprising the amino acidsequence shown in SEQ ID NO: 3. Further homologous α-amylases include anα-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB12512, NCIB 12513 or DSM 9375, all of which are described in detail inWO 95/26397, and the α-amylase described by Tsukamoto et al.,Biochemical and Biophysical Research Communications, 151 (1988), pp.25-31. Also the novel α-amylase of the invention, of which a specificembodiment is shown in SEQ ID NO: 24 (and SEQ ID NO: 26), iscontemplated as the parent α-amylase to be mutated according to theinvention.

Still further homologous α-amylases include the α-amylase produced bythe B. licheniformis strain described in EP 0252666 (ATCC 27811), andthe α-amylases identified in WO 91/00353 and WO 94/18314. Othercommercial Termamyl-like B. licheniformis α-amylases are Duramyl™ fromNovo Nordisk, Optitherm™ and Takatherm™ (available from Solvay),Maxamyl™ (available from Gist-brocades/Genencor), Spezym AA™ and SpezymeDelta AA™ (available from Genencor), and Keistase™ (available fromDaiwa).

Because of the substantial homology found between these α-amylases, theyare considered to belong to the same class of α-amylases, namely theclass of “Termamyl-like α-amylases”.

Accordingly, in the present context, the term “Termamyl-like α-amylase”is intended to indicate an α-amylase which, at the amino acid level,exhibits a substantial homology to Termamyl™ , i.e., the B.licheniformis α-amylase having the amino acid sequence shown in SEQ IDNO: 4 herein. In other words, a Termamyl-like α-amylase is an α-amylasewhich has the amino acid sequence shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6,7 or 8 herein, and the amino acid sequence shown in SEQ ID NO: 1 of WO95/26397 (the same as the amino acid sequence shown as SEQ ID NO: 7herein) or in SEQ ID NO: 2 of WO 95/26397 (the same as the amino acidsequence shown as SEQ ID NO: 8 herein) or in Tsukamoto et al., 1988,(which amino acid sequence is shown in SEQ ID NO: 6 herein) or i) whichdisplays at least 60%, preferred at least 70%, more preferred at least75%, even more preferred at least 80%, especially at least 85%,especially preferred at least 90%, even especially more preferred atleast 95% homology with at least one of said amino acid sequences shownin SEQ ID NOS: 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 and/or ii) displaysimmunological cross-reactivity with an antibody raised against at leastone of said α-amylases, and/or iii) is encoded by a DNA sequence whichhybridizes to the DNA sequences encoding the above-specified α-amylaseswhich are apparent from SEQ ID NOS: 9, 10, 11, 12 or 13 of the presentapplication (which encoding sequences encode the amino acid sequencesshown in SEQ ID NOS: 1, 2, 3, 4 and 5 herein, respectively), from SEQ IDNO: 4 of WO 95/26397 (which DNA sequence, together with the stop codonTAA, is shown in SEQ ID NO: 14 herein and encodes the amino acidsequence shown in SEQ ID NO: 8 herein) and from SEQ ID NO: 5 of WO95/26397 (shown in SEQ ID NO: 15 herein), respectively.

In connection with property i), the “homology” may be determined by useof any conventional algorithm, preferably by use of the GAP progammefrom the GCG package version 7.3 (June 1993) using default values forGAP penalties, which is a GAP creation penalty of 3.0 and GAP extensionpenalty of 0.1, (Genetic Computer Group (1991) Programme Manual for theGCG Package, version 7, 575 Science Drive, Madison, Wis., USA 53711).

A structural alignment between Termamyl and a Termamyl-like α-amylasemay be used to identify equivalent/corresponding positions in otherTermamyl-like α-amylases. One method of obtaining said structuralalignment is to use the Pile Up programme from the GCG package usingdefault values of gap penalties, i.e., a gap creation penalty of 3.0 andgap extension penalty of 0.1. Other structural alignment methods includethe hydrophobic cluster analysis (Gaboriaud et al., (1987), FEBS LETTERS224, pp. 149-155) and reverse threading (Huber, T; Torda, AE, PROTEINSCIENCE Vol. 7, No. 1 pp. 142-149 (1998).

Property ii) of the α-amylase, i.e., the immunological cross reactivity,may be assayed using an antibody raised against, or reactive with, atleast one epitope of the relevant Termamyl-like α-amylase. The antibody,which may either be monoclonal or polyclonal, may be produced by methodsknown in the art, e.g. as described by Hudson et al., PracticalImmunology, Third edition (1989), Blackwell Scientific Publications. Theimmunological cross-reactivity may be determined using assays known inthe art, examples of which are Western Blotting or radialimmunodiffusion assay, e.g., as described by Hudson et al., 1989. Inthis respect, immunological cross-reactivity between the α-amylaseshaving the amino acid sequences SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, or 8respectively, have been found.

The oligonucleotide probe used in the characterization of theTermamyl-like α-amylase in accordance with property iii) above maysuitably be prepared on the basis of the full or partial nucleotide oramino acid sequence of the α-amylase in question.

Suitable conditions for testing hybridization involve presoaking in5×SSC and prehybridizing for 1 hour at ˜40° C. in a solution of 20%formamide, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50mg of denatured sonicated calf thymus DNA, followed by hybridization inthe same solution supplemented with 100 mM ATP for 18 hours at ˜40° C.,followed by three times washing of the filter in 2×SSC, 0.2% SDS at 40°C. for 30 minutes (low stringency), preferred at 50° C. (mediumstringency), more preferably at 65° C. (high stringency), even morepreferably at ˜75° C. (very high stringency). More details about thehybridization method can be found in Sambrook et al., Molecular Cloning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.

In the present context, “derived from” is intended not only to indicatean α-amylase produced or producible by a strain of the organism inquestion, but also an α-amylase encoded by a DNA sequence isolated fromsuch strain and produced in a host organism transformed with said DNAsequence. Finally, the term is intended to indicate an α-amylase whichis encoded by a DNA sequence of synthetic and/or cDNA origin and whichhas the identifying characteristics of the α-amylase in question. Theterm is also intended to indicate that the parent α-amylase may be avariant of a naturally occurring α-amylase, i.e., a variant which is theresult of a modification (insertion, substitution, deletion) of one ormore amino acid residues of the naturally occurring α-amylase.

Parent Hybrid α-amylases

The parent α-amylase may be a hybrid α-amylase, i.e., an α-amylase whichcomprises a combination of partial amino acid sequences derived from atleast two α-amylases.

The parent hybrid α-amylase may be one which on the basis of amino acidhomology and/or immunological cross-reactivity and/or DNA hybridization(as defined above) can be determined to belong to the Termamyl-likeα-amylase family. In this case, the hybrid α-amylase is typicallycomposed of at least one part of a Termamyl-like α-amylase and part(s)of one or more other α-amylases selected from Termamyl-like α-amylasesor non-Termamyl-like α-amylases of microbial (bacterial or fungal)and/or mammalian origin.

Thus, the parent hybrid α-amylase may comprise a combination of partialamino acid sequences deriving from at least two Termamyl-likeα-amylases, or from at least one Termamyl-like and at least onenon-Termamyl-like bacterial α-amylase, or from at least oneTermamyl-like and at least one fungal α-amylase. The Termamyl-likeα-amylase from which a partial amino acid sequence derives may, e.g., beany of those specific Termamyl-like α-amylases referred to herein.

For instance, the parent α-amylase may comprise a C-terminal part of anα-amylase derived from a strain of B. licheniformis, and a N-terminalpart of an α-amylase derived from a strain of B. amyloliquefaciens orfrom a strain of B. stearothermophilus. For instance, the parentα-amylase may comprise at least 430 amino acid residues of theC-terminal part of the B. licheniformis α-amylase, and may, e.g.,comprise a) an amino acid segment corresponding to the 37 N-terminalamino acid residues of the B. amyloliquefaciens α-amylase having theamino acid sequence shown in SEQ ID NO: 5 and an amino acid segmentcorresponding to the 445 C-terminal amino acid residues of the B.licheniformis α-amylase having the amino acid sequence shown in SEQ IDNO: 4, or b) an amino acid segment corresponding to the 68 N-terminalamino acid residues of the B. stearothermophilus α-amylase having theamino acid sequence shown in SEQ ID NO: 3 and an amino acid segmentcorresponding to the 415 C-terminal amino acid residues of the B.licheniformis α-amylase having the amino acid sequence shown in SEQ IDNO: 4.

The non-Termamyl-like α-amylase may, e.g., be a fungal α-amylase, amammalian or a plant α-amylase or a bacterial α-amylase (different froma Termamyl-like α-amylase). Specific examples of such α-amylases includethe Aspergillus oryzae TAKA α-amylase, the A. niger acid α-amylase, theBacillus subtilis α-amylase, the porcine pancreatic α-amylase and abarley α-amylase. All of these α-amylases have elucidated structureswhich are markedly different from the structure of a typicalTermamyl-like α-amylase as referred to herein.

The fungal α-amylases mentioned above, i.e., derived from A. niger andA. oryzae, are highly homologous on the amino acid level and generallyconsidered to belong to the same family of α-amylases. The fungalα-amylase derived from Aspergillus oryzae is commercially availableunder the tradename Fungamyl™.

Furthermore, when a particular variant of a Termamyl-like α-amylase(variant of the invention) is referred to—in a conventional manner—byreference to modification (e.g. deletion or substitution) of specificamino acid residues in the amino acid sequence of a specificTermamyl-like α-amylase, it is to be understood that variants of anotherTermamyl-like α-amylase modified in the equivalent position(s) (asdetermined from the best possible amino acid sequence alignment betweenthe respective amino acid sequences) are encompassed thereby.

A preferred embodiment of a variant of the invention is one derived froma B. licheniformis α-amylase (as parent Termamyl-like α-amylase), e.g.,one of those referred to above, such as the B. licheniformis α-amylasehaving the amino acid sequence shown in SEQ ID NO: 4.

Construction of Variants of the Invention

The construction of the variant of interest may be accomplished bycultivating a microorganism comprising a DNA sequence encoding thevariant under conditions which are conducive for producing the variant.The variant may then subsequently be recovered from the resultingculture broth. This is described in detail further below.

Altered Properties of Variants of the Invention

The following discusses the relationship between mutations which may bepresent in variants of the invention, and desirable alterations inproperties (relative to those a parent, Termamyl-like α-amylase) whichmay result therefrom.

Increased Thermostability at Acidic pH and/or at Low Ca²⁺ Concentration

Mutations of particular relevance in relation to obtaining variantsaccording to the invention having increased thermostability at acidic pHand/or at low Ca²⁺ concentration include mutations at the followingpositions (relative to B. licheniformis α-amylase, SEQ ID NO: 4): H156,N172, A181, N188, N190, H205, D207, A209, A210, E211, Q264, N265.

In the context of the invention the term “acidic pH” means a pH below7.0, especially below the pH range, in which industrial starchliquefaction processes are normally performed, which is between pH 5.5and 6.2.

In the context of the present invention the term “low Calciumconcentration” means concentrations below the normal level used inindustrial starch liquefaction. Normal concentrations vary depending ofthe concentration of free Ca²⁺ in the corn. Normally a dosagecorresponding to 1 mM (40 ppm) is added which together with the level incorn gives between 40 and 60 ppm free Ca²⁺.

In the context of the invention the term “high tempertatures” meanstemperatures between 95° C. and 160° C., especially the temperaturerange in which industrial starch liquefaction processes are normallyperformed, which is between 95° C. and 105° C.

The inventors have now found that the thermostability at acidic pHand/or at low Ca²⁺ concentration may be increased even more by combiningcertain mutations including the above mentioned mutations and/or I201with each other.

Said “certain” mutations are the following (relative to B. licheniformisα-amylase, SEQ ID NO: 4): N190, D207, E211, Q264 and I201.

Said mutation may further be combined with deletions in one, preferablytwo or even three positions as described in WO 96/23873 (i.e., inpositions R181, G182, T183, G184 in SEQ ID NO: 1 herein). According tothe invention variants of a parent Termamyl-like α-amylase withα-amylase activity comprising mutations in two, three, four, five or sixof the above positions are contemplated.

It should be emphazised that not only the Termamyl-like α-amylasesmentioned specifically below are contemplated. Also other commercialTermamyl-like α-amylases are contemplated. An unexhaustive list of suchα-amylases is the following:

α-amylases produced by the B. licheniformis strain described in EP0252666 (ATCC 27811), and the α-amylases identified in WO 91/00353 andWO 94/18314. Other commercial Termamyl-like B. licheniformis α-amylasesare Optitherm™ and Takatherm™ (available from Solvay), Maxamyl™(available from Gist-brocades/Genencor), Spezym AA™ Spezyme Delta AA™(available from Genencor), and Keistase™ (available from Daiwa).

It may be mentioned here that amino acid residues, respectively, atpositions corresponding to N190, I201, D207 and E211, respectively, inSEQ ID NO: 4 constitute amino acid residues which are conserved innumerous Termamyl-like α-amylases. Thus, for example, the correspondingpositions of these residues in the amino acid sequences of a number ofTermamyl-like α-amylases which have already been mentioned (vide supra)are as follows:

TABLE 1 Termamyl-like α-amylase N I D E Q B. licheniformis (SEQ ID NO:4) N190 I201 D207 E211 Q264 B. amyloliquefaciens (SEQ ID NO: 5) N190V201 D207 E211 Q264 B. stearothermophilus (SEQ ID N193 L204 E210 E214 —NO: 3) Bacillus WO 95/26397 (SEQ ID N195 V206 E212 E216 — NO: 2)Bacillus WO 95/26397 (SEQ ID N195 V206 E212 E216 — NO: 1) “Bacillus sp.#707” (SEQ ID NO: 6) N195 I206 E212 E216 — Bacillus sp. AA560 (SEQ IDNO: 24) N195 I206 E212 E216 —

Mutations of these conserved amino acid residues are very important inrelation to improving thermostability at acidic pH and/or at low calciumconcentration, and the following mutations are of particular interest inthis connection (with reference to the numbering of the B. licheniformisamino acid sequence shown in SEQ ID NO: 4).

Pair-wise amino acid deletions at positions corresponding to R179-G182in SEQ ID NO: 5 corresponding to a gap in SEQ ID NO: 4. when alignedwith a numerous Termamyl-like α-amylases. Thus, for example, thecorresponding positions of these residues in the amino acid sequences ofa number of Termamyl-like α-amylases which have already been mentioned(vide supra) are as follows:

TABLE 2 Pair wise amino Termamyl-like α-amylase acid deletions among B.amyloliquefaciens (SEQ ID NO: 5) R176, G177, E178, G179 B.stearothermophilus (SEQ ID NO: 3) R179, G180, I181, G182 Bacillus WO95/26397 (SEQ ID NO: 2) R181, G182, T183, G184 Bacilius WO 95/26397 (SEQID NO: 1) R181, G182, D183, G184 “Bacillus sp. #707” (SEQ ID NO: 6)R181, G182, H183, G184 Bacillus sp. (AA560) (SEQ ID NO: 24) R181, G182,H183, G184

When using SEQ ID NO: 1-6 or SEQ ID NO: 24 (or SEQ ID NO: 26) as thebackbone (i.e., as the parent Termamyl-like α-amylase) two, three, four,five or six mutations may according to the invention be made in thefollowing regions/positions to increase the thermostability at acidic pHand/or at low Ca²⁺ concentrations (relative to the parent):

(relative to SEQ ID NO: 1 herein):

1: R181*, G182*, T183*, G184*

2: N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

3: V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

4: E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

5: E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

(relative to SEQ ID NO: 2 herein):

1: R181*,G182*,D183*,G184*

2: N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

3: V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

4: E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

5: E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

(Relative to SEQ ID NO: 3 herein):

1: R179*,G180,I181*,G182*

2: N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

3: L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;

4: E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

5: E214A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

6: S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V

Relative to SEQ ID NO: 4 herein):

1: Q178*,G179*

2: N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

3: I201A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

4: D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

5: E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

6: Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

(relative to SEQ ID NO: 5 herein):

1: R176*,G177*,E178,G179*

2: N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

3: V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

4: D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

5: E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

6: Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

(relative to SEQ ID NO: 6 herein):

1: R181*,G182*,H183*,G184*

2: N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

3: I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

4: E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

5: E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V; and

(relative to SEQ ID NO: 24)

1: R191*,G182*,H183*,G184*

2: N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

3: I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

4: E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

5: E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V.

Contemplated according to the present invention is combining three,four, five or six mutation.

Specific double mutations for backbone SEQ ID NO: 1-6 and SEQ ID NO: 24and SEQ ID NO: 26 are listed in the following.

Using SEQ ID NO: 1 as the backbone the following double mutations arecontemplated according to the invention:

—R181*/G182*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G182*/T183*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—T183*/G184*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R181*/G182*/V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

—G182*/T183*/V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

—T183*/G184*/V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

—R181*/G182*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G182*/T183*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—T183*/G184*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V:

—R181*/G182*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G182*/T183*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—T183*/G184*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R181*/G182*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—G182*/T183*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—T183*/G184*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

—N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y

/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

Using SEQ ID NO: 2 as the backbone the following double mutations arecontemplated according to the invention:

—R181*/G182*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G182*/D183*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—D183*/G184*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R181*/G182*/V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

—G182*/T183*/V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

—T183*/G184*/V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

—R181*/G182*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G182*/T183*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—T183*/G184*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R181*/G182*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G182*/T183*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—T183*/G184*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R181*/G182*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—G182*/T183*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—T183*/G184*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

—N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y

/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

Using SEQ ID NO: 3 as the backbone the following double mutations arecontemplated according to the invention:

—R179*/G180*/N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G180*/I181*/N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—I181*/G182*/N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R179*/G180*/L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;

—G180*/I181*/L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;

—I181*/G182*/L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;

—R179*/G180*/E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G180*/I181*/E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—I181*/G182*/E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R179*/G180*/E214A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G180*/I181*/E214A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—I181*/G182*/E214A,P,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R179*/G180*/S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

—G180*/I181*/S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

—I181*/G182*/S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

—N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;

—N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E214A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

—L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V

/E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V

/E214A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V

/S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

—E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

—E214A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

Using SEQ ID NO: 4 as the backbone the following double mutations arecontemplated according to the invention:

—Q178*/G179*/N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—Q178*/G179*/I201A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

—Q178*/G179*/D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—Q178*/G179*/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R179*/G180*/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—N190/I201A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

—N190/D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—N190/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—N190/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—I201/D207A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—I201/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—I201/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—D207/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—D207/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—E211/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Using SEQ ID NO: 5 as the backbone the following double mutations arecontemplated according to the invention:

—R176*/G177*/N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G177*/E178*/N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—E178*/G179*/N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R176*/G177*/V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

—G176*/E178*/V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

—E178*/G179*/V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

—R176*/G177*/D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G177*/E178*/D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—E178*/G179*/D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R176*/G177*/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G177*/E178*/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—E178*/G179*/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R176*/G177*/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G177*/E178*/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—E178*/G179*/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

—N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y

/D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y

/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y

/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

Using SEQ ID NO: 6 as the backbone the following double mutations arecontemplated according to the invention:

—R181*/G182*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G182*/H183*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—H183*/G184*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R181*/G182*/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

—G182*/H183*/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

—H183*/G184*/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

—R181*/G182*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G182*/H183*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—H183*/G184*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R181*/G182*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G182*/H183*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—H183*/G184*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R181*/G182*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—G182*/H183*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—H183*/G184*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

—N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V

/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—I206A,P,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V.

Using SEQ ID NO: 24 as the backbone the following double Imutantions arecontemplated according to the invention:

—R181*/G182*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G182*/H183*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—H183*/G184*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R181*/G182*/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

—G182*/H183*/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

—H183*/G184*/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

—R181*/G182*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G182*/H183*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—H183*/G184*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R181*/G182*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—G182*/H183*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—H183*/G184*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—R181*/G182*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—G182*/H183*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—H183*/G184*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

—N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V

/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

—E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

—E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V.

All Termamyl-like α-amylase defined above may suitably be used asbackbone for preparing variants of the invention.

However, in a preferred embodiment the variant comprises the followingmutations: N190F/Q264S in SEQ ID NO: 4 or in corresponding positiones inanother parent Termamyl-like α-amylases.

In another embodiment the variant of the invention comprises thefollowing mutations: I181*/G182*/N193F in SEQ ID NO: 3 (TVB146) or incorresponding positions in another parent Termamyl-like α-amylases. Saidvariant may further comprise a substitution in position E214Q.

In a preferred embodiment of the invention the parent Termamyl-likeα-amylase is a hybrid α-amylase of SEQ ID NO: 4 and SEQ ID NO: 5.Specifically, the parent hybrid Termamyl-like α-amylase may be a hybridalpha-amylase comprising the 445 C-terminal amino acid residues of theB. licheniformis α-amylase shown in SEQ ID NO: 4 and the 37 N-terminalamino acid residues of the α-amylase derived from B. amyloliquefaciensshown in SEQ ID NO: 5, which may suitably further have the followingmutations: H156Y+A181T+N190E+A209V+Q264S (using the numbering in SEQ IDNO: 4). The latter mentioned hybrid is used in the examples below and isreferred to as LE174.

General Mutations of the Invention

It may be preferred that a variant of the invention comprises one ormore modifications in addition to those outlined above. Thus, it may beadvantageous that one or more proline residues present in the part ofthe α-amylase variant which is modified is/are replaced with anon-proline residue which may be any of the possible, naturallyoccurring non-proline residues, and which preferably is an alanine,glycine, serine, threonine, valine or leucine.

Analogously, it may be preferred that one or more cysteine residuespresent among the amino acid residues with which the parent α-amylase ismodified is/are replaced with a non-cysteine residue such as serine,alanine, threonine, glycine, valine or leucine.

Furthermore, a variant of the invention may—either as the onlymodification or in combination with any of the above outlinedmodifications—be modified so that one or more Asp and/or Glu present inan amino acid fragment corresponding to the amino acid fragment 185-209of SEQ ID NO: 4 is replaced by an Asn and/or Gln, respectively. Also ofinterest is the replacement, in the Termamyl-like α-amylase, of one ormore of the Lys residues present in an amino acid fragment correspondingto the amino acid fragment 185-209 of SEQ ID NO: 4 by an Arg.

It will be understood that the present invention encompasses variantsincorporating two or more of the above outlined modifications.

Furthermore, it may be advantageous to introduce point-mutations in anyof the variants described herein.

Mutations with may suitably made include mutationes in the followingpositions: H133, M15, M197, A209.

Cloning a DNA Sequence Encoding an α-amylase

The DNA sequence encoding a parent α-amylase may be isolated from anycell or microorganism producing the α-amylase in question, using variousmethods well known in the art. First, a genomic DNA and/or cDNA libraryshould be constructed using chromosomal DNA or messenger RNA from theorganism that produces the α-amylase to be studied. Then, if the aminoacid sequence of the α-amylase is known, homologous, labelledoligonucleotide probes may be synthesized and used to identifyα-amylase-encoding clones from a genomic library prepared from theorganism in question. Alternatively, a labelled oligonucleotide probecontaining sequences homologous to a known α-amylase gene could be usedas a probe to identify α-amylase-encoding clones, using hybridizationand washing conditions of lower stringency.

Yet another method for identifying α-amylase-encoding clones wouldinvolve inserting fragments of genomic DNA into an expression vector,such as a plasmid, transforming α-amylase-negative bacteria with theresulting genomic DNA library, and then plating the transformed bacteriaonto agar containing a substrate for α-amylase, thereby allowing clonesexpressing the α-amylase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g. the phosphoroamiditemethod described by S. L. Beaucage and M. H. Caruthers (1981) or themethod described by Matthes et al. (1984). In the phosphoroamiditemethod, oligonucleotides are synthesized, e.g., in an automatic DNAsynthesizer, purified, annealed, ligated and cloned in appropriatevectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate, the fragments corresponding to various parts of the entireDNA sequence), in accordance with standard techniques. The DNA sequencemay also be prepared by polymerase chain reaction (PCR) using specificprimers, for instance as described in U.S. Pat. No. 4,683,202 or R. K.Saiki et al. (1988).

Site-directed Mutagenesis

Once an α-amylase-encoding DNA sequence has been isolated, and desirablesites for mutation identified, mutations may be introduced usingsynthetic oligonucleotides. These oligonucleotides contain nucleotidesequences flanking the desired mutation sites; mutant nucleotides areinserted during oligonucleotide synthesis. In a specific method, asingle-stranded gap of DNA, bridging the α-amylase-encoding sequence, iscreated in a vector carrying the α-amylase gene. Then the syntheticnucleotide, bearing the desired mutation, is annealed to a homologousportion of the single-stranded DNA. The remaining gap is then filled inwith DNA polymerase I (Klenow fragment) and the construct is ligatedusing T4 ligase. A specific example of this method is described inMorinaga et al. (1984). U.S. Pat. No. 4,760,025 discloses theintroduction of oligonucleotides encoding multiple mutations byperforming minor alterations of the cassette. However, an even greatervariety of mutations can be introduced at any one time by the Morinagamethod, because a multitude of oligonucleotides, of various lengths, canbe introduced.

Another method for introducing mutations into α-amylase-encoding DNAsequences is described in Nelson and Long (1989). It involves the 3-stepgeneration of a PCR fragment containing the desired mutation introducedby using a chemically synthesized DNA strand as one of the primers inthe PCR reactions. From the PCR-generated fragment, a DNA fragmentcarrying the mutation may be isolated by cleavage with restrictionendonucleases and reinserted into an expression plasmid.

Random Mutagenesis

Random mutagenesis is suitably performed either as localised orregion-specific random mutagenesis in at least three parts of the genetranslating to the amino acid sequence shown in question, or within thewhole gene.

The random mutagenesis of a DNA sequence encoding a parent α-amylase maybe conveniently performed by use of any method known in the art.

In relation to the above, a further aspect of the present inventionrelates to a method for generating a variant of a parent α-amylase, e.g.wherein the variant exhibits altered or increased thermal stabilityrelative to the parent, the method comprising:

(a) subjecting a DNA sequence encoding the parent α-amylase to randommutagenesis,

(b) expressing the mutated DNA sequence obtained in step (a) in a hostcell, and

(c) screening for host cells expressing an α-amylase variant which hasan altered property (i.e. thermal stability) relative to the parentα-amylase.

Step (a) of the above method of the invention is preferably performedusing doped primers.

For instance, the random mutagenesis may be performed by use of asuitable physical or chemical mutagenizing agent, by use of a suitableoligonucleotide, or by subjecting the DNA sequence to PCR generatedmutagenesis. Furthermore, the random mutagenesis may be performed by useof any combination of these mutagenizing agents. The mutagenizing agentmay, e.g., be one which induces transitions, transversions, inversions,scrambling, deletions, and/or insertions.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) ir-radiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues. When such agents are used, themutagenesis is typically performed by incubating the DNA sequenceencoding the parent enzyme to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions for themutagenesis to take place, and selecting for mutated DNA having thedesired properties.

When the mutagenesis is performed by the use of an oligonucleotide, theoligonucleotide may be doped or spiked with the three non-parentnucleotides during the synthesis of the oligonucleotide at the positionswhich are to be changed. The doping or spiking may be done so thatcodons for unwanted amino acids are avoided. The doped or spikedoligonucleotide can be incorporated into the DNA encoding the α-amylaseenzyme by any published technique, using e.g. PCR, LCR or any DNApolymerase and ligase as deemed appropriate.

Preferably, the doping is carried out using “constant random doping”, inwhich the percentage of wild-type and mutation in each position ispredefined. Furthermore, the doping may be directed toward a preferencefor the introduction of certain nucleotides, and thereby a preferencefor the introduction of one or more specific amino acid residues. Thedoping may be made, e.g., so as to allow for the introduction of 90%wild type and 10% mutations in each position. An additionalconsideration in the choice of a doping scheme is based on genetic aswell as protein-structural constraints. The doping scheme may be made byusing the DOPE program which, inter alia, ensures that introduction ofstop codons is avoided.

When PCR-generated mutagenesis is used, either a chemically treated ornon-treated gene encoding a parent α-amylase is subjected to PCR underconditions that increase the mis-incorporation of nucleotides (Deshler1992; Leung et al., Technique, Vol. 1, 1989, pp. 11-15).

A mutator strain of E. coli (Fowler et al., Molec. Gen. Genet., 133,1974, pp. 179-191), S. cereviseae or any other microbial organism may beused for the random mutagenesis of the DNA encoding the α-amylase by,e.g., transforming a plasmid containing the parent glycosylase into themutator strain, growing the mutator strain with the plasmid andisolating the mutated plasmid from the mutator strain. The mutatedplasmid may be subsequently transformed into the expression organism.

The DNA sequence to be mutagenized may be conveniently present in agenomic or cDNA library prepared from an organism expressing the parentα-amylase. Alternatively, the DNA sequence may be present on a suitablevector such as a plasmid or a bacteriophage, which as such may beincubated with or other-wise exposed to the mutagenising agent. The DNAto be mutagenized may also be present in a host cell either by beingintegrated in the genome of said cell or by being present on a vectorharboured in the cell. Finally, the DNA to be mutagenized may be inisolated form. It will be understood that the DNA sequence to besubjected to random mutagenesis is preferably a cDNA or a genomic DNAsequence.

In some cases it may be convenient to amplify the mutated DNA sequenceprior to performing the expression step b) or the screening step c).Such amplification may be performed in accordance with methods known inthe art, the presently preferred method being PCR-generatedamplification using oligonucleotide primers prepared on the basis of theDNA or amino acid sequence of the parent enzyme.

Subsequent to the incubation with or exposure to the mutagenising agent,the mutated DNA is expressed by culturing a suitable host cell carryingthe DNA sequence under conditions allowing expression to take place. Thehost cell used for this purpose may be one which has been transformedwith the mutated DNA sequence, optionally present on a vector, or onewhich was carried the DNA sequence encoding the parent enzyme during themutagenesis treatment. Examples of suitable host cells are thefollowing: gram positive bacteria such as Bacillus subtilis, Bacilluslicheniformnis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium, Bacillus thuringiensis, Streptomyces lividans orStreptomyces murinus; and gram-negative bacteria such as E. coli.

The mutated DNA sequence may further comprise a DNA sequence encodingfunctions permitting expression of the mutated DNA sequence.

Localized Random Mutagenesis

The random mutagenesis may be advantageously localized to a part of theparent α-amylase in question. This may, e.g., be advantageous whencertain regions of the enzyme have been identified to be of particularimportance for a given property of the enzyme, and when modified areexpected to result in a variant having improved properties. Such regionsmay normally be identified when the tertiary structure of the parentenzyme has been elucidated and related to the function of the enzyme.

The localized, or region-specific, random mutagenesis is convenientlyperformed by use of PCR generated mutagenesis techniques as describedabove or any other suitable technique known in the art. Alternatively,the DNA sequence encoding the part of the DNA sequence to be modifiedmay be isolated, e.g., by insertion into a suitable vector, and saidpart may be subsequently subjected to mutagenesis by use of any of themutagenesis methods discussed above.

Alternative Methods of Providing α-amylase Variants

Alternative methods for providing variants of the invention include geneshuffling method known in the art including the methods e.g. describedin WO 95/22625 (from Affymax Technologies N.V.) and WO 96/00343 (fromNovo Nordisk A/S).

Expression of α-amylase Variants

According to the invention, a DNA sequence encoding the variant producedby methods described above, or by any alternative methods known in theart, can be expressed, in enzyme form, using an expression vector whichtypically includes control sequences encoding a promoter, operator,ribosome binding site, translation initiation signal, and, optionally, arepressor gene or various activator genes.

The recombinant expression vector carrying the DNA sequence encoding anα-amylase variant of the invention may be any vector which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. Thus, the vector may be an autonomously replicating vector,i.e. a vector which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g. aplasmid, a bacteriophage or an extrachromosomal element, minichromosomeor an artificial chromosome. Alternatively, the vector may be one which,when introduced into a host cell, is integrated into the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated.

In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Examples of suitable promoters for directing thetranscription of the DNA sequence encoding an α-amylase variant of theinvention, especially in a bacterial host, are the promoter of the lacoperon of E. coli, the Streptomyces coelicolor agarase gene dagApromoters, the promoters of the Bacillus licheniformis α-amylase gene(amyL), the promoters of the Bacillus stearothermophilus maltogenicamylase gene (amyM), the promoters of the Bacillus amyloliquefaciensα-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylBgenes etc. For transcription in a fungal host, examples of usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralα-amylase, A. niger acid stable α-amylase, A. niger glucoamylase,Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triosephosphate isomerase or A. nidulans acetamidase.

The expression vector of the invention may also comprise a suitabletranscription terminator and, in eukaryotes, polyadenylation sequencesoperably connected to the DNA sequence encoding the α-amylase variant ofthe invention. Termination and polyadenylation sequences may suitably bederived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question. Examples of such sequences arethe origins of replication of plasmids pUC19, pACYC177, pUB110, pE194,pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g., as described in WO 91/17243.

While intracellular expression may be advantageous in some respects,e.g., when using certain bacteria as host cells, it is generallypreferred that the expression is extracellular. In general, the Bacillusα-amylases mentioned herein comprise a preregion permitting secretion ofthe expressed protease into the culture medium. If desirable, thispreregion may be replaced by a different preregion or signal sequence,conveniently accomplished by substitution of the DNA sequences encodingthe respective preregions.

The procedures used to ligate the DNA construct of the inventionencoding an α-amylase variant, the promoter, terminator and otherelements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989).

The cell of the invention, either comprising a DNA construct or anexpression vector of the invention as defined above, is advantageouslyused as a host cell in the recombinant production of an α-amylasevariant of the invention. The cell may be transformed with the DNAconstruct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g. by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

The cell of the invention may be a cell of a higher organism such as amammal or an insect, but is preferably a microbial cell, e.g. abacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are grampositive bacteria such as Bacillussubtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis,Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamryloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gramnegative bacteria such as E.coli. The transformation of the bacteria may, for instance, be effectedby protoplast transformation or by using competent cells in a mannerknown per se.

The yeast organism may favourably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g., Saccharornyces cerevisiae.The filamentous fungus may advantageously belong to a species ofAspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Fungal cellsmay be transformed by a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known per se. A suitable procedure for transformationof Aspergillus host cells is described in EP 238 023.

In yet a further aspect, the present invention relates to a method ofproducing an α-amylase variant of the invention, which method comprisescultivating a host cell as described above under conditions conducive tothe production of the variant and recovering the variant from the cellsand/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in question and obtaining expressionof the α-amylase variant of the invention. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedrecipes (e.g. as described in catalogues of the American Type CultureCollection).

The α-amylase variant secreted from the host cells may conveniently berecovered from the culture medium by well-known procedures, includingseparating the cells from the medium by centrifugation or filtration,and precipitating proteinaceous components of the medium by means of asalt such as ammonium sulphate, followed by the use of chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

Compositions

In a still further aspect, the present invention relates to compositionscomprising an α-amylase or α-amylase variant of the present invention.Preferably, the compositions are enriched in an α-amylase or α-amylasevariant of the present invention. In the present context, the term“enriched” indicates that the α-amylase activity of the composition hasbeen increased, e.g., with an enrichment factor of 1.1.

The composition may comprise an α-amylase or α-amylase variant of theinvention as the major enzymatic component, e.g., a mono-componentcomposition. Alternatively, the composition may comprise multipleenzymatic activities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be producible by means of amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus niger, or Aspergillusoryzae, or Trichoderma, Humicola, preferably Humicola insolens, orFusarium, preferably Fusarium bactridioides, Fusarium cerealis, Fusariumcrookwellense, Fusarium culmorum, Fusarium grarminearum, Fusariumgraminum, Fusarium heterosporum, Fusarium negundi, Fusariumr oxwysporum,Fusariurn reticulaturn, Fusarium roseurm, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusariumrtrichothecioides, or Fusarium venenatum.

The α-amylase compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the α-amylase composition may be in the formof a granulate or a microgranulate. The polypeptide to be included inthe composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the α-amylase compositionsof the invention. The dosage of the α-amylase composition of theinvention and other conditions under which the composition is used maybe determined on the basis of methods known in the art.

Further Compositions

The invention also relates to a composition comprising a mixture of oneor more α-amylase or α-amylase variant of the invention derived from (asthe parent Termamyl-like α-amylase) the B. stearothermophilus α-amylasehaving the sequence shown in SEQ ID NO: 3 and a Termamyl-likealpha-amylase derived from the B. licheniformis α-amylase having thesequence shown in SEQ ID NO: 4.

Further, the invention also relates to a composition comprising amixture of one or more variants according the invention derived from (asthe parent Termamyl-like α-amylase) the B. stearothermophilus α-amylasehaving the sequence shown in SEQ ID NO: 3 and a hybrid alpha-amylasecomprising a part of the B. amryloliquefaciens α-amylase shown in SEQ IDNO: 5 and a part of the B. licheniformis α-amylase shown in SEQ ID NO:4. The latter mentioned hydrid Termamyl-like α-amylase comprises the 445C-terminal amino acid residues of the B. licheniformis α-amylase shownin SEQ ID NO: 4 and the 37 N-terminal amino acid residues of theα-amylase derived from B. amryloliquefaciens shown in SEQ ID NO: 5. Saidlatter mentioned hybrid α-amylase may suitably comprise the followingmutations: H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ IDNO: 4). In the examples below said hybrid parent Termamyl-likeα-amylase, is used in combination with variants of the invention, whichvariants may be used in compositions of the invention.

In a specific embodiment of the invention the composition comprises amixture of TVB146 and LE174, e.g., in a ratio of 2:1 to 1:2, such as1:1.

An α-amylase or α-amylase variant of the invention or a composition ofthe invention may in an aspect of the invention be used for washingand/or dishwashing; for textile desizing or for starch liquefaction.

Detergent Compositions

The α-amylase or α-amylase variant of the invention may be added to andthus become a component of a detergent composition.

The detergent composition of the invention may for example be formulatedas a hand or machine laundry detergent composition including a laundryadditive composition suitable for pre-treatment of stained fabrics and arinse added fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additivecomprising the enzyme of the invention. The detergent additive as wellas the detergent composition may comprise one or more other enzymes suchas a protease, a lipase, a cutinase, an amylase, a carbohydrase, acellulase, a pectinase, a mannanase, an arabinase, a galactanase, axylanase, an oxidase, e.g., a laccase, and/or a peroxidase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metallo protease, preferably an alkaline microbialprotease or a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g., of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and274.

Preferred commercially available protease enzymes include Alcalase®,Savinase®, Primase®, Duralase®, Esperase®, and Kannase® (Novo NordiskA/S), Maxatase®, Maxacal, Maxapem®, Properase®, Purafect®, PurafectOxP®, FN2®, and FN3® (Genencor International Inc.).

Lipases: Suitable lipases include those of bac-terial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes(EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g.,from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta,1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Preferred commercially available lipase enzymes include Lipolase™ andLipolase Ultra™ (Novo Nordisk A/S).

Amylases: Suitable amylases (α- and/or β-) include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Amylases include, for example, α-amylases obtained fromBacillus, e.g., a special strain of B. licheniformis, described in moredetail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ andBAN™ (Novo Nordisk A/S), Rapidase™ and Purastar™ (from GenencorInterna-tional Inc.).

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulose variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299.

Commercially available cellulases include Celluzyme®, and Carezyme®(Novo Nordisk A/S), Clazinase®, and Puradax HA® (Genencor InternationalInc.), and KAC-500(B)® (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bac-terial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme®, (Novo NordiskA/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated e.g, as a granulate, a liquid, a slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonyl-phenol ethoxylate, alkylpolyglycoside, alkyldimethylamine-oxide,ethoxylated fatty acid monoethanol-amide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, tripho-sphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetri-aminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

The detergent may contain a bleaching system which may comprise a H2O2source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxyben-zenesul-fonate. Alternatively, the bleaching system maycomprise peroxyacids of e.g. the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the com-position may be formulated as described in, e.g., WO92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g. fabric conditioners in-cluding clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions anyenzyme, in particular the enzyme of the invention, may be added in anamount corresponding to 0.01-100 mg of enzyme protein per liter of washliqour, preferably 0.05-5 mg of enzyme protein per liter of wash liqour,in particular 0.1-1 mg of enzyme protein per liter of wash liqour.

The enzyme of the invention may additionally be incorporated in thedetergent formulations disclosed in WO 97/07202 which is herebyincorporated as reference

Further Detergent Compositions

As mentioned above, variants of the invention may suitably beincorporated in detergent compositions. Increased thermostability at lowcalcium concentrations would be very beneficial for amylase performancein detergents, i.e., the alkaline region. Reference is made, forexample, to WO 96/23874 and WO 97/07202 for further details concerningrelevant ingredients of detergent compositions (such as laundry ordishwashing detergents), appropriate methods of formulating the variantsin such detergent compositions, and for examples of relevant types ofdetergent compositions.

Detergent compositions comprising an α-amylase or α-amylase variant ofthe invention may additionally comprise one or more other enzymes, suchas a lipase, cutinase, protease, cellulase, peroxidase or laccase,and/or another α-amylase.

An α-amylase or α-amylase variant of the invention may be incorporatedin detergents at conventionally employed concentrations. It is atpresent contemplated that a variant of the invention may be incorporatedin an amount corresponding to 0.00001-1 mg (calculated as pure, activeenzyme protein) of α-amylase per liter of wash/dishwash liquor usingconventional dosing levels of detergent.

Dishwash Deterget Compositions

The α-amyiase or α-amylase variant of the invention may also be used indishwash detergent compositions, including the following:

1) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant 0.4-2.5%Sodium metasilicate  0-20% Sodium disilicate  3-20% Sodium triphosphate20-40% Sodium carbonate  0-20% Sodium perborate 2-9% Tetraacetylethylene diamine (TAED) 1-4% Sodium sulphate  5-33% Enzymes0.0001-0.1%  

2) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant (e.g.alcohol ethoxylate) 1-2% Sodium disilicate  2-30% Sodium carbonate10-50% Sodium phosphonate 0-5% Trisodium citrate dihydrate  9-30%Nitrilotrisodium acetate (NTA)  0-20% Sodium perborate monohydrate 5-10% Tetraacetyl ethylene diamine (TAED) 1-2% Polyacrylate polymer(e.g. maleic acid/acrylic acid  6-25% copolymer) Enzymes 0.0001-0.1%  Perfume 0.1-0.5% Water 5-10 

3) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant 0.5-2.0%Sodium disilicate 25-40% Sodium citrate 30-55% Sodium carbonate  0-29%Sodium bicarbonate  0-20% Sodium perborate monohydrate  0-15%Tetraacetyl ethylene diamine (TAED) 0-6% Maleic acid/acrylic acidcopolymer 0-5% Clay 1-3% Polyamino acids  0-20% Sodium polyacrylate 0-8%Enzymes 0.0001-0.1%  

4) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant 1-2%Zeolite MAP 15-42% Sodium disilicate 30-34% Sodium citrate  0-12% Sodiumcarbonate  0-20% Sodium perborate monohydrate  7-15% Tetraacetylethylene diamine (TAED) 0-3% Polymer 0-4% Maleic acid/acrylic acidcopolymer 0-5% Organic phosphonate 0-4% Clay 1-2% Enzymes 0.0001-0.1%  Sodium sulphate Balance

5) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant 1-7%Sodium disilicate 18-30% Trisodium citrate 10-24% Sodium carbonate12-20% Monopersulphate (2 KHSO₅.KHSO₄.K₂SO₄) 15-21% Bleach stabilizer0.1-2%   Maleic acid/acrylic acid copolymer 0-6% Diethylene triaminepentaacetate, pentasodium salt   0-2.5% Enzymes 0.0001-0.1%   Sodiumsulphate, water Balance

6) POWDER AND LIQUID DISHWASHING COMPOSITION WITH CLEANING SURFACTANTSYSTEM Nonionic surfactant   0-1.5% Octadecyl dimethylamine N-oxidedihydrate 0-5% 80:20 wt. C18/916 blend of octadecyl dimethylamine 0-4%N-oxide dihydrate and hexadecyldimethyl amine N-oxide dihydrate 70:30wt. C18/C16 blend of octadecyl bis(hydroxy- 0-5% ethyl)amine N-oxideanhydrous and hexadecyl bis(hydroxyethyl)amine N-oxide anhydrous C₁₃-C₁₅alkyl ethoxysulfate with an average degree of  0-10% ethoxylation of 3C₁₂-C₁₅ alkyl ethoxysulfate with an average degree of 0-5% ethoxylationof 3 C₁₃-C₁₅ ethoxylated alcohol with an average degree of 0-5%ethoxylation of 12 A blend of C₁₂-C₁₅ ethoxylated alcohols with an  0-6.5% average degree of ethoxylation of 9 A blend of C₁₃-C₁₅ethoxylated alcohols with an 0-4% average degree of ethoxylation of 30Sodium disilicate  0-33% Sodium tripolyphosphate  0-46% Sodium citrate 0-28% Citric acid  0-29% Sodium carbonate  0-20% Sodium perboratemonohydrate   0-11.5% Tetraacetyl ethylene diamine (TAED) 0-4% Maleicacid/acrylic acid copolymer   0-7.5% Sodium sulphate   0-12.5% Enzymes0.0001-0.1%  

7) NON-AQUEOUS LIQUID AUTOMATIC DISHWASHING COMPOSITION Liquid nonionicsurfactant (e.g. alcohol ethoxylates)  2.0-10.0% Alkali metal silicate 3.0-15.0% Alkali metal phosphate 20.0-40.0% Liquid carrier selectedfrom higher glycols, poly- 25.0-45.0% glycols, polyoxides, glycolethersStabilizer (e.g. a partial ester of phosphoric acid and a 0.5-7.0%C₁₆-C₁₈ alkanol) Foam suppressor (e.g. silicone)   0-1.5% Enzymes0.0001-0.1%  

8) NON-AQUEOUS LIQUID DISHWASHING COMPOSITION Liquid nonionic surfactant(e.g. alcohol ethoxylates) 2.0-40.0% Sodium silicate 3.0-15.0% Alkalimetal carbonate 7.0-20.0% Sodium citrate 0.0-1.5%  Stabilizing system(e.g. mixtures of finely divided 0.5-7.0%  silicone and low molecularweight dialkyl polyglycol ethers) Low molecule weight polyacrylatepolymer 5.0-15.0% Clay gel thickener (e.g. bentonite) 0.0-10.0%Hydroxypropyl cellulose polymer 0.0-0.6%  Enzymes 0.0001-0.1%   Liquidcarrier selected from higher lycols, poly- Balance glycols, polyoxidesand glycol ethers

9) THIXOTROPIC LIQUID AUTOMATIC DISHWASHING COMPOSITION C₁₂-C₁₄ fattyacid 0-0.5% Block co-polymer surfactant 1.5-15.0%   Sodium citrate0-12%  Sodium tripolyphosphate 0-15%  Sodium carbonate 0-8%   Aluminiumtristearate 0-0.1% Sodium cumene sulphonate 0-1.7% Polyacrylatethickener 1.32-2.5%   Sodium polyacrylate 2.4-6.0%   Boric acid 0-4.0%Sodium formate  0-0.45% Calcium formate 0-0.2% Sodium n-decydiphenyloxide disulphonate 0-4.0% Monoethanol amine (MEA)  0-1.86% Sodiumhydroxide (50%) 1.9-9.3%   1,2-Propanediol 0-9.4% Enzymes 0.0001-0.1%   Suds suppressor, dye, perfumes, water Balance

10) LIQUID AUTOMATIC DISHWASHING COMPOSITION Alcohol ethoxylate 0-20%Fatty acid ester sulphonate 0-30% Sodium dodecyl sulphate 0-20% Alkylpolyglycoside 0-21% Oleic acid 0-10% Sodium disilicate monohydrate18-33%  Sodium citrate dihydrate 18-33%  Sodium stearate  0-2.5% Sodiumperborate monohydrate 0-13% Tetraacetyl ethylene diamine (TAED) 0-8% Maleic acid/acrylic acid copolymer 4-8%  Enzymes 0.0001-0.1%  

11) LIQUID AUTOMATIC DISHWASHER COMPOSITION CONTAINING PROTECTED BLEACHPARTICLES Sodium silicate  5-10% Tetrapotassium pyrophosphate 15-25%Sodium triphosphate 0-2% Potassium carbonate 4-8% Protected bleachparticles, e.g. chlorine  5-10% Polymeric thickener 0.7-1.5% Potassiumhydroxide 0-2% Enzymes 0.0001-0.1%   Water Balance

11) Automatic dishwashing compositions as described in 1), 2) 3), 4), 6)and 10), wherein perborate is replaced by percarbonate.

12) Automatic dishwashing compositions as described in 1)-6) whichadditionally contain a manganese catalyst. The manganese catalyst may,e.g., be one of the compounds described in “Efficient manganesecatalysts for low-temperature bleaching”, Nature 369, 1994, pp. 637-639.

Uses

The present invention is also directed to methods for using an α-amylaseor α-amylase variant of the invention in detergents, in particularlaundry detergent compositions and dishwash detergent compositions.

Industrial Applications

An α-amylase and α-amylase varaint of the invention are well suited foruse in a variety of industrial processes, in particular the enzymes ofthe invention finds potential applications as a component in detergents,e.g., laundry, dishwash and hard surface cleaning detergentcompositions, but it may also be useful in the production of sweetenersand ethanol from starch. Thus, it may be used in conventionalstarch-converting processes, such as liquefaction and saccharificationprocesses described in U.S. Pat. No. 3,912,590 and EP patentpublications Nos. 252,730 and 63,909.

An α-amylase or α-amylase variant of the invention may also be used inthe production of lignocellulosic materials, such as pulp, paper andcardboard, from starch reinforced waste paper and cardboard, especiallywhere repulping occurs at pH above 7 and where amylases can facilitatethe disintegration of the waste material through degradation of thereinforcing starch. The α-amylase of the invention is especially usefulin a process for producing a papermaking pulp from starch-coated printedpaper. The process may be performed as described in WO 95/14807,comprising the following steps:

a) disintegrating the paper to produce a pulp,

b) treating with a starch-degrading enzyme before, during or after stepa), and

c) separating ink particles from the pulp after steps a) and b).

An α-amylase or α-amylase variant of the invention may also be veryuseful in modifying starch where enzymatically modified starch is usedin papermaking together with alkaline fillers such as calcium carbonate,kaolin and clays. With the alkaline α-amylases of the invention itbecomes possible to modify the starch in the presence of the filler thusallowing for a simpler integrated process.

An α-amylase or α-amylase variant of the invention may also be veryuseful in textile desizing. In the textile processing industry,α-amylases are traditionally used as auxiliaries in the desizing processto facilitate the removal of starch-containing size which has served asa protective coating on weft yarns during weaving. Complete removal ofthe size coating after weaving is import-ant to ensure optimum resultsin the subsequent processes, in which the fabric is scoured, bleachedand dyed. Enzymatic starch break-down is preferred because it does notinvolve any harmful effect on the fiber material. In order to reduceprocessing cost and increase mill through-put, the desizing processingis sometimes combined with the scouring and bleaching steps. In suchcases, non-enzymatic auxiliaries such as alkali or oxidation agents aretypically used to break down the starch, because traditional α-amylasesare not very compatible with high pH levels and bleaching agents. Thenon-enzymatic breakdown of the starch size does lead to some fiberdamage because of the rather aggressive chemicals used. Accordingly, itwould be desirable to use the α-amylases of the invention as they havean improved performance in alkaline solutions. The α-amylases may beused alone or in combination with a cellulase when desizingcellulose-containing fabric or textile.

The α-amylases of the invention may also be very useful in a beer-makingprocess; the α-amylases will typically be added during the mashingprocess.

Production of Sweeteners from Starch

A “traditional” process for conversion of starch to fructose syrupsnormally consists of three consecutive enzymatic processes, viz., aliquefaction process followed by a saccharification process and anisomerization process. During the liquefaction process, starch isdegraded to dextrins by an α-amylase (e.g., Termamyl™) at pH valuesbetween 5.5 and 6.2 and at temperatures of 95-160° C. for a period ofapprox. 2 hours. In order to ensure an optimal enzyme stability underthese conditions, 1 mM of calcium is added (40 ppm free calcium ions).

After the liquefaction process the dextrins are converted into dextroseby addition of a glucoamylase (e.g. AMG™) and a debranching enzyme, suchas an isoamylase or a pullulanase (e.g. Promozyme™). Before this stepthe pH is reduced to a value below 4.5, maintaining the high temperature(above 95° C.), and the liquefying α-amylase activity is denatured. Thetemperature is lowered to 60° C., and glucoamylase and debranchingenzyme are added. The saccharification process proceeds for 24-72 hours.

After the saccharification process the pH is increased to a value in therange of 6-8, preferably pH 7.5, and the calcium is removed by ionexchange. The dextrose syrup is then converted into high fructose syrupusing, e.g., an immmobilized glucoseisomerase (such as Sweetzyme™).

At least 1 enzymatic improvements of this process could be envisaged.Reduction of the calcium dependency of the liquefying α-amylase.Addition of free calcium is required to ensure adequately high stabilityof the α-amylase, but free calcium strongly inhibits the activity of theglucoseisomerase and needs to be removed, by means of an expensive unitoperation, to an extent which reduces the level of free calcium to below3-5 ppm. Cost savings could be obtained if such an operation could beavoided and the liquefaction process could be performed without additionof free calcium ions.

To achieve that, a less calcium-dependent Termamyl-like α-amylase whichis stable and highly active at low concentrations of free calcium (<40ppm) is required. Such a Termamyl-like α-amylase should have a pHoptimum at a pH in the range of 4.5-6.5, preferably in the range of4.5-5.5.

MATERIALS AND METHODS

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Enzymes:

SP690: α-amylase shown in SEQ ID NO: 1

SP722: α-amylase shown in SEQ ID NO: 2

Termamyl®: α-amylase from Bacillus licheniformis shown in SEQ ID NO: 4.

AA560: α-amylase of the invention shown in SEQ ID NO: 24 encoded by theDNA sequence shown in SEQ ID NO: 23.

AA360: α-amylase shown in SEQ ID NO: 26 being identical to the AA560α-amylase encoded by the DNA sequence shown in SEQ ID NO: 25.

BSG alpha-amylase: B. stearothermophilus alpha-amylase depicted in SEQID NO: 3.

TVB146 alpha-amylase variant: B. stearothermophilus alpha-amylasevariant depicted in SEQ ID NO: 3 with the following mutations: with thedeletion in positions I181−G182+N193F.

LE174 hybrid alpha-amylase variant:

LE174 is a hybrid Termamyl-like alpha-amylase being identical to theTermamyl sequence, i.e., the Bacillus licheniformis α-amylase shown inSEQ ID NO: 4, except that the N-terminal 35 amino acid residues (of themature protein) has been replaced by the N-terminal 33 residues of BAN(mature protein), i.e., the Bacillus amyloliquefaciens alpha-amylaseshown in SEQ ID NO: 5, which further have the following mutations:H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 4).LE174 was constructed by SOE-PCR (Higuchi et al. 1988, Nucleic AcidsResearch 16:7351).

Model Detergent:

A/P (Asia/Pacific) Model Detergent has the following composition: 20%STPP (sodium tripolyphosphate), 25% Na₂SO₄, 15% Na₂CO₃, 20% LAS (linearalkylbenzene sulfonate, Nansa 80S), 5% C₁₂-C₁₅, alcohol ethoxylate(Dobanol 25-7), 5% Na₂Si₂O₅, 0.3% NaCl. Omo Multi Acao (Brazil), Omoconcentrated powder (Europe) (product of Unilever) Ariel Futur liquid(Europe) (product of Procter and Gamble)

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Deutshe Sammmlung von Microorganismen undZellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig DE,and given the following accession number:

Deposit Accession Number Date of Deposit NN017557 DSM 12648 25 January1999 NN017560 DSM 12649 25 January 1999 NN049467 DSM12761 7^(th) April1999 NN049470 DSM12764 7^(th) April 1999

The strains have been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Host Organism

Bacillus subtilis strain SHa273 is disclosed in WO 95/10603 E. colistrain SJ2 (Diderichsen et al. (1990)).

Plasmids:

The gene bank vector pSJ1678 is disclosed in WO 94/19454 which is herebyincorporated by reference.

pTVB110 is a plasmid replicating in Bacillus subtilis by the use oforigin of replication from pUB110 (Gryczan, T. J. (1978) J. Bact.134:318-329). The plasmid further encodes the cat gene, conferringresistance towards chlorampenicol, obtained from plasmid pC194(Horinouchi, S. and Weisblum, B. (1982), J. Bact. 150: 815-825). Theplasmid harbors a truncated version of the Bacillus licheniformisα-amylase gene, amyL, such that the amyL promoter, signal sequence andtranscription terminator are present, but the plasmid does not providean amy-plus phenotype (halo formation on starch containing agar).

Methods

General Molecular Biology Methods:

Unless otherwise mentioned the DNA manipulations and transformationswere performed using standard methods of molecular biology (Sambrook etal. (1989); Ausubel et al. (1995); Harwood and Cutting (1990).

Fermentation and Purification of α-amylase Variants

Fermentation may be performed by methods well known in the art or asfollows.

A B. subtilis strain harbouring the relevant expression plasmid isstreaked on a LB-agar plate with 10 μg/ml kanamycin from −80° C. stock,and grown overnight at 37° C. The colonies are transferred to 100 ml BPXmedia supplemented with 10 μg/ml kanamycin in a 500 ml shaking flask.Composition of BPX medium:

Potato starch 100 g/l Barley flour 50 g/l BAN 5000 SKB 0.1 g/l Sodiumcaseinate 10 g/l Soy Bean Meal 20 g/l Na₂HPO₄, 12 H₂O 9 g/l Pluronic TM0.1 g/l

The culture is shaken at 37° C. at 270 rpm for 5 days.

Cells and cell debris are removed from the fermentation broth bycentrifugation at 4500 rpm in 20-25 minutes. Afterwards the supernatantis filtered to obtain a completely clear solution. The filtrate isconcentrated and washed on a UF-filter (10000 cut off membrane) and thebuffer is changed to 20 mM Acetate pH 5.5. The UF-filtrate is applied ona S-sepharose F.F. and elution is carried out by step elution with 0.2MNaCl in the same buffer. The eluate is dialysed against 10 mM Tris, pH9.0 and applied on a Q-sepharose F.F. and eluted with a linear gradientfrom 0-0.3M NaCl over 6 column volumes. The fractions which contain theactivity (measured by the Phadebas assay) are pooled, pH was adjusted topH 7.5 and remaining color was removed by a treatment with 0.5% W/vol.active coal in 5 minutes.

ASSAYS FOR DETERMINING α-AMYLASE ACTIVITY

Activity Determination—(KNU)

One Kilo alpha-amylase Unit (1 KNU) is the amount of enzyme which breaksdown 5.26 g starch (Merck, Amylum Solubile, Erg. B 6, Batch 9947275) perhour in Novo Nordisk's standard method for determination ofalpha-amylase based upon the following condition:

Substrate soluble starch Calcium content in solvent 0.0043 M Reactiontime 7-20 minutes Temperature 37° C. pH 5.6

Detailed description of Novo Nordisk's analytical method (AF 9) isavailable on request.

BS-amylase Activity Determination—KNU(S)

1. Application Field

This method is used to determine α-amylase activity in fermentation andrecovery samples and formulated and granulated products.

2. Principle

BS-amylase breaks down the substrate(4,6-ethylidene(G₇)-p-nitrophenyl(G₁)-α,D-maltoheptaoside (written asethylidene-G₇-PNP) into, among other things, G₂-PNP and G₃-PNP, where Gdenoted glucose and PNP p-nitrophenol.

G2-PNP and G3-PNP are broken down by α-glucosidase, which is added inexcess, into glucose and the yellow-coloured p-nitrophenol.

The colour reaction is monitored in situ and the change in absorbanceover time calculated as an expression of the spreed of the reaction andthus of the activity of the enzyme. See the Boehringer Mannheim 1442 309guidelines for further details.

2.1 Reaction conditions Reaction: Temperature : 37° C. pH : 7.1Pre-incubation time : 2 minutes Detection: wavelength : 405 nmMeasurement time : 3 minutes

3. Definition of Units

Bacillus stearothermophius alpha-amylase (BS-amylase) activity isdetermined relative to a standard of declared activity and stated inKilo Novo Units (Stearothermophilus) or KNU(S)).

4. Specificity and Sensitivity

Limit of determination: approx. 0.4 KNU(s)/g

5. Apparatus

Cobas Fara analyser

Diluted (e.g. Hamilton Microlab 1000)

Analytical balance (e.g. Mettler AE 100)

Stirrer plates

6. Reagents/Substrates

A ready-made kit is used in this analysis to determine α-amylaseactivity. Note that the reagents specified for the substrate andα-glucosidase are not used as described in the Boehringer Mannheimguidelines. However, the designations “buffer”, “glass 1″, glass 1a″ andGlass 2″ are those referred to in those guidelines.

6.1. Substrate

4,6-ethylidene(G₇)-p-nitrophenyl(G₁)-α,D-maltoheptaoside (written asethylidene-G₇-PNP) e.g. Boehringer Mannheim 1442 309

6.2 α-glucosidase Help Reagent

α-glucosidase, e.g. Boehringer Mannheim 1442 309

6.3 BRIJ 35 solution BRIJ 3 (30% W/V Sigma 430 AG-6) 1000 mLDemineralized water up to 2,000 mL 6.4 Stabiliser Brij 35 solution 33 mLCaCl₂*2 H₂O (Merck 2382) 882 g Demineralized water up to 2,000 mL

7. Samples and Standards

7.1 Standard Curve

Example: Preparation of BS-amylase standard curve

The relevant standard is diluted to 0.60 KNU(s)/mL as follows. Acalculated quantity of standard is weighed out and added to 200 mLvolumetric flask, which is filled to around the ⅔ mark withdemineralized water. Stabiliser corresponding to 1% of the volume of theflask is added and the flask is filled to the mark with demineralizedwater.

A Hamilton Microlab 1000 is used to produce the dilutions shown below.Demineralized water with 1% stabiliser is used as the diluent.

Enzyme stock Dilution No. solution 1% stabiliser KNU (s)/mL 1 20 μL 580μL 0.02 2 30 μL 570 μL 0.03 3 40 μL 560 μL 0.04 4 50 μL 550 μL 0.05 5 60μL 540 μL 0.06

7.2 Level Control

A Novo Nordisk A/S BS amylase level control is included in all runsusing the Cobas Fara. The control is diluted with 1% stabiliser so thatthe final dilution is within the range of the standard curve. Allweights and dilutions are noted on the worklist

7.3 Sample Solutions

Single Determination

Fermentation samples (not final samples) from production, allfermentation samples from pilot plants and storage stability samples areweighed out and analyzed once only.

Double Determination Over 1 Run:

Process samples, final fermentation samples from production, samplesfrom GLP studies and R&D samples are weighed out and analyzed twice.

Double Determinations Over 2 Runs:

Finished product samples are weighed out and analyzed twice over twoseparate runs.

Maximum concentration of samples in powder form: 5%

Test samples are diluted with demineralized water with 1% stabiliser toapprox. 0.037 KNU(S)/mL on the basis of their expected activity. Thefinal dilution is made direct into the sample cup.

8. Procedure

8.1 Cobas Menu Program

The Cobas Menu Program is used to suggest the weight/dilutions ofsamples and level control to be used.

The samples are entered into the program with a unique identificationcode and a worklist is printed out

The samples and control are weighed out and diluted as stated on theworklist with hand-written weight data is inserted into the BS-amylaseanalysis logbook

The results are computered automatically by the Cobas Fara as describedin item 9 and printed out along with the standard curve.

Worklists and results printouts are inserted into the BS-amylaseanalysis logbook.

8.2 Cobas Fara set-up

The samples are placed in the sample rack

The five standards are placed in the calibration rack at position 1 to 5(strongest standard at position 5), and control placed in the same rackat position 10.

The substrate is transferred to a 30 mL reagent container and placed inthat reagent rack at position 2 (holder 1).

The α-glucosidase help reagent is transferred to a 50 mL reagentcontainer and placed in the reagent rack at position 2 (holder C)

8.3 Cobas Fare analysis

The main principles of the analysis are as follows:

20 μL sample and 10 μL rinse-water are pipetted into the cuvette alongwith 250 μL α-glucosidase help reagent. The cuvette rotates for 10seconds and the reagents are thrown out into the horizontal cuvettes. 25μL substrate and 20 μL rinse-water are pipetted off. After a 1 secondwait to ensure that the temperature is 37° C., the cuvette rotates againand the substrate is mixed into the horizontal cuvettes. Absorbance ismeasured for the first time after 120 seconds and then every 5 seconds.Absorbance is measured a total of 37 times for each sample.

9. Calculations

The activity of the samples is calculated relative to Novo Nordisk A/Sstandard.

The standard curve is plotted by the analyzer. The curve is to be gentlycurved, rising steadily to an absorbance of around 0.25 for standard no.5.

The activity of the samples in KNU(S)/mL is read off the standard curveby the analyzer.

The final calculations to allow for the weights/dilutions used employthe following formula:

Activity in KNU(S)/g=S×V×F/W

S=analysis result read off (KNU(S)/mL

V=volume of volumetric flask used in mL

F=dilution factor for second dilution

W=weight of enzyme sample in g

9.2 Calculation of Mean Values

Results are stated with 3 significant digits. However, for sampleactivity<10 KNU(S)/g, only 2 significant digits are given.

The following rules apply on calculation of mean values:

1. Data which deviates more than 2 standard deviations from the meanvalue is not included in the calculation.

2. Single and double determination over one run: The mean value iscalculated on basis of results lying within the standard curve'sactivity area.

3. Double determinations over two runs: All values are included in themean value. Outliers are omitted.

10. Accuracy and Precision

The coefficient of variation is 2.9% based on retrospective validationof analysis results for a number of finished products and the levelcontrol.

Phadebas Assay (for α-amylase activity determination)

α-amylase activity is determined by a method employing Phadebas® tabletsas substrate. Phadebas tablets (Phadebas® Amylase Test, supplied byPharmacia Diagnostic) contain a cross-linked insoluble blue-coloredstarch polymer which has been mixed with bovine serum albumin and abuffer substance and tabletted.

For every single measurement one tablet is suspended in a tubecontaining 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mMphosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂, pH adjusted to thevalue of interest with NaOH). The test is performed in a water bath atthe temperature of interest. The α-amylase to be tested is diluted in xml of 50 mM Britton-Robinson buffer. 1 ml of this α-amylase solution isadded to the 5 ml 50 mM Britton-Robinson buffer. The starch ishydrolyzed by the α-amylase giving soluble blue fragments. Theabsorbance of the resulting blue solution, measuredspectrophotometrically at 620 nm, is a function of the α-amylaseactivity.

It is important that the measured 620 nm absorbance after 10 or 15minutes of incubation (testing time) is in the range of 0.2 to 2.0absorbance units at 620 nm. In this absorbance range there is linearitybetween activity and absorbance (Lambert-Beer law). The dilution of theenzyme must therefore be adjusted to fit this criterion. Under aspecified set of conditions (temp., pH, reaction time, bufferconditions) 1 mg of a given α-amylase will hydrolyze a certain amount ofsubstrate and a blue colour will be produced. The colour intensity ismeasured at 620 nm. The measured absorbance is directly proportional tothe specific activity (activity/mg of pure α-amylase protein) of theα-amylase in question under the given set of conditions.

Alternative α-amylase Activity Method (PNP-G7 assay)

α-amylase activity is determined by a method employing the PNP-G7substrate. PNP-G7 which is a abbreviation forp-nitrophenyl-α,D-maltoheptaoside is a blocked oligosaccharide which canbe cleaved by an endo-amylase. Following the cleavage, the α-Glucosidaseincluded in the kit digest the substrate to liberate a free PNP moleculewhich has a yellow colour and thus can be measured by visiblespectophometry at λ=405 nm. (400-420 nm.). Kits containing PNP-G7substrate and α-Glucosidase is manufactured by Boehringer-Mannheim(cat.No. 1054635).

To prepare the substrate one bottle of substrate (BM 1442309) is addedto 5 ml buffer (BM1442309). To prepare the α-Glucosidase one bottle ofα-Glucosidase (BM 1462309) is added to 45 ml buffer (BM1442309). Theworking solution is made by mixing 5 ml α-Glucosidase solution with 0.5ml substrate.

The assay is performed by transforming 20 μl enzyme solution to a 96well microtitre plate and incubating at 25° C. 200 μl working solution,25° C. is added. The solution is mixed and pre-incubated 1 minute andabsorption is measured every 15 sec. over 3 minutes at OD 405 nm.

The slope of the time dependent absorption-curve is directlyproportional to the specific activity (activity per mg enzyme) of theα-amylase in question under the given set of conditions.

EXAMPLES Example 1

Construction of Variants of BSG α-amylase (SEQ ID NO: 3)

The gene encoding BSG, amyS, is located in plasmid pPL1117. This plasmidcontains also the gene conferring resistance towards kanamycin and anorigin of replication, both obtained from plasmid pUB110 (Gryczan, T. J.et al (1978) J. Bact 134:318-329).

The DNA sequence of the mature part of amyS is shown as SEQ ID NO: 11and the amino acid sequence of the mature protein is shown as SEQ ID NO:3

BSG variant TVB145, which contains a deletion of 6 nucleotidescorresponding to amino acids I181-G182 in the mature protein, isconstructed as follows:

Polymerase Chain Reaction (PCR) is utilized to amplify the part of theamyS gene (from plasmid pPL1117), located between DNA primers BSG1 (SEQID NO: 16) and BSGM2 (SEQ ID NO: 19). BSG1 is identical to a part of theamyS gene whereas BSGM2 contains the 6 bp nucleotide deletion. Astandard PCR reaction is carried out: 94° C. for 5 minutes, 25 cycles of(94° C. for 45 seconds, 50° C. for 45 seconds, 72° C. for 90 seconds),72° C. for 7 minutes using the Pwo polymerase under conditions asrecommended by the manufacturer, Boehringer Mannheim Gmbh.

The resulting approximately 550 bp amplified band was used as amegaprimer (Barik, S and Galinski, MS (1991): Biotechniques 10: 489-490)together with primer BSG3 in a second PCR with pPL1117 as templateresulting in a DNA fragment of approximately 1080 bp.

This DNA fragment is digested with restriction endonucleases Acc65I andSalI and the resulting approximately 550 bp fragment is ligated intoplasmid pPL1117 digested with the same enzymes and transformed into theprotease- and amylase-deleted Bacillus subtilis strain SHA273 (describedin WO 92/11357 and WO 95/10603).

Kanamycin resistant and starch degrading transformants were analysed forthe presence of the desired mutations (restriction digest to verify theintroduction of a HindIII site in the gene). The DNA sequence betweenrestriction sites Acc65I and SalI was verified by DNA sequencing toensure the presence of only the desired mutations.

BSG variant TVB146 which contains the same 6 nucleotide deletion asTVB145 and an additional substitution of asparagine 193 for aphenylalanine, N193F, was constructed in a similar way as TVB145utilizing primer BSGM3 (SEQ ID NO: 20) in the first PCR.

BSG variant TVB161, containing the deletion of I181-G182, N193F, andL204F, is constructed in a similar way as the two previous variantsexcept that the template for the PCR reactions is plasmid pTVB146(pPL1117 containing the TVB146-mutations within amyS and the mutagenicoligonucleotide for the first PCR is BSGM3.

BSG variant TVB162, containing the deletion of I181-G182, N193F, andE210H, is constructed in a similar way as TVB161 except that themutagenic oligonucleotide is BSGM4 (SEQ ID NO: 21).

BSG variant TVB163, containing the deletion of I181-G182, N193F, andE214Q, is constructed in a similar way as TVB161 except that themutagenic oligonucleotide is BSGM5 (SEQ ID NO: 22).

The above constructed BSG variants were then fermented and purified asdescribed above in the “Material and Methods” section.

Example 2

Measurement of the Calcium- and pH-dependent Stability

Normally, the industrial liquefaction process runs using pH 6.0-6.2 asliquefaction pH and an addition of 40 ppm free calcium in order toimprove the stability at 95° C.-105° C. Some of the herein proposedsubstitutions have been made in order to improve the stability at

1. lower pH than pH 6.2 and/or

2. at free calcium levels lower than 40 ppm free calcium.

Two different methods have been used to measure the improvements instability obtained by the different substitutions in the α-amylase fromB. stearothermophilus:

Method 1. One assay which measures the stability at reduced pH, pH 5.0,in the presence of 5 ppm free calcium. 10 μg of the variant wereincubated under the following conditions: A 0.1 M acetate solution, pHadjusted to pH 5.0, containing 5 ppm calcium and 5% w/w common cornstarch (free of calcium). Incubation was made in a water bath at 95° C.for 30 minutes.

Method 2. One assay which measure the stability in the absence of freecalcium and where the pH is maintained at pH 6.0. This assay measuresthe decrease in calcium sensitivity: 10 μg of the variant were incubatedunder the following conditions: A 0.1 M acetate solution, pH adjusted topH 6.0, containing 5% w/w common corn starch (free of calcium).Incubation was made in a water bath at 95° C. for 30 minutes.

Stability Determination

All the stability trials 1, 2 have been made using the same set up. Themethod was:

The enzyme was incubated under the relevant conditions (1-4). Sampleswere taken at 0, 5, 10, 15 and 30 minutes and diluted 25 times (samedilution for all taken samples) in assay buffer (0.1M 50 mM Brittonbuffer pH 7.3) and the activity was measured using the Phadebas assay(Pharmacia) under standard conditions pH 7.3, 37° C.

The activity measured before incubation (0 minutes) was used asreference (100%). The decline in percent was calculated as a function ofthe incubation time. The table shows the residual activity after 30minutes of incubation.

Stability method 1./Low pH stability improvement SEQ. ID SEQ. ID NO: 3SEQ. ID NO: 3 VARIANT NO: 3 VARIANT WITH VARIANT WITH DELETION WITHDELETION IN POS. WT. SEQ. DELETION IN POS. I181-G182 + MINUTES ID. NO: 3IN POS. I181-G182 + N193F + OF AMYLASE I181-G182 N193F E214Q INCUBATION(BSG) (TVB145) (TVB146) (TVB163) 0 100 100 100 100 5 29 71 83 77 10 9 6277 70 15 3 50 72 67 30 1 33 62 60

Stability method 1./Low pH stability improvement The temperaturedescribet in method 1 has been reduced from 95° C. to 70° C. since theamylases mentioned for SEQ ID NO: 1 and

SEQ. ID SEQ. ID NO: 2 NO: 1 VARIANT VARIANT WITH WITH MINUTES WT. SEQ.DELETION SEQ. ID DELETION OF ID. NO: 2 IN POS. NO: 1 IN POS. INCUBATIONAMYLASE D813-G184 AMYLASE T183-G184 0 100 100 100 100 5 73 92 41 76 1059 88 19 69 15 48 91 11 62 30 28 92 3 59

Stability method 2./Low pH stability improvement SEQ. ID SEQ. ID NO: 3SEQ. ID NO: 3 VARIANT NO: 3 VARIANT WITH VARIANT WITH DELETION WITHDELETION IN POS. WT. SEQ. DELETION IN POS. I181-G182 + MINUTES ID. NO: 3IN POS. I181-G182 + N193F + OF AMYLASE I181-G182 N193F E214Q INCUBATION(BSG) (TVB145) (TVB146) (TVB163) 0 100 100 100 100 5 60 82 81 82 10 4276 80 83 15 31 77 81 79 30 15 67 78 79

Specific Activity Determination.

The specific activity was determined using the Phadebas assay(Pharmacia) as activity/mg enzyme. The activity was determined using theα-amylase assay described in the Materials and Methods section herein.

The specific activity of the parent enzyme and a single and a doublemutation was determined to:

BSG: SEQ ID NO:3 (Parent enzyme) 20000 NU/mg TVB145: SEQ ID NO:3 withthe deletion in positions I181-G182: (Single mutation) 34600 NU/mgTVB146: SEQ ID NO:3 with the deletion in positions I181-G182 + N193E:(Double mutation) 36600 NU/mg TVB163: SEQ ID NO:3 with the deletion inpositions I181-G182 + N193E + E214Q: (Triple mutation) 36300 NU/mg

Example 3

Pilot Plant Jet Cook and Liquefaction with Alpha-amylase Variant TVB146

Pilot plant liquefaction experiments were run in the mini-jet systemusing a dosage of 50 NU (S)/g DS at pH 5.5 with 5 ppm added Ca⁺⁺, tocompare the performance of formulated BSG alpha-amylase variant TVB146(SEQ ID NO: 3 with deletion in positions I181−G182+N193F) with that ofparent BSG alpha-amylase (SEQ ID NO: 3). The reaction was monitored bymeasuring the DE increase (Neocuproine method) as a function of time.

Corn starch slurries were prepared by suspending 11.8 kg CerestarC*Pharm GL 03406 (89% starch) in deionized water and making up to 30 kg.The pH was adjusted to 5.5 at ambient temperature, after the addition of0.55 g CaCl₂. 2H₂O.

The following enzymes were used:

TVB146 108 KNU(S)/g, 146 KNU(SM9)/g BSG amylase 101 KNU(S)/g, 98KNU(SM9)/g

An amount of enzyme corresponding to 50 NU (SM9)/g DS was added, and theconductivity adjusted to 300 mS using NaCl. The standard conditions wereas follows:

Substrate concentration 35% w/w (initial)

31.6-31.9% w/w (final)

Temperature 105° C., 5 minutes (Primary liquefaction) 95° C., 90 minutes(Secondary liquefaction) pH (initial) 5.5

After jetting, the liquefied starch was collected and transported insealed thermos-flasks from the pilot plant to the laboratory, wheresecondary liquefaction was continued at 95° C.

10 ml samples were taken at 15 minute intervals from 15-90 minutes. 2drops of 1 N HCl were added to inactivate the enzyme. From thesesamples, 0.3-0.1 g (according to the expected DE) were weighed out anddiluted to 100 ml. Reducing sugars were then determined according to theNeocuproine method (Determination of reducing sugar with improvedprecision. Dygert, Li, Florida and Thomas (1965). Anal. Biochem 13, 368)and DE values determined. The development of DE as a function of time isgiven in the following table:

Time TVB146 BSG (min.) DE (neocuproine) 15 2.80 2.32 30 4.88 3.56 456.58 4.98 60 8.17 6.00 75 9.91 7.40 90 11.23 8.03

As can be seen the alpha-amylase variant TB146 performed significantlybetter under industrially relevant application conditions at low levelsof calcium than the parent BSG alpha-amylase.

Example 4

Jet Cook and Liquefaction with a Combination of Alpha-amylase Variants(TVB146 and LE174)

Jet cook and liquefaction using a combination of the alpha-amylasevariants, TVB146 and LE174 (ratio 1:1) were carried out at the followingconditions:

Substrate A.E. Staley food grade powdered corn starch (100 lbs)

D.S. 35% using DI water

Free Ca²⁺ 2.7 ppm at pH 5.3 (none added, from the starch only)

Initial pH 5.3

Dose AF9 units (AF9 is available on request) for each enzyme variant was28 NU/g starch db for a total dose of 56 NU/g

Temperature in primary liquefaction 105° C.

Hold time in primary liquefaction 5 minutes

Temperature in secondary liquefaction 95° C.

At 15 minutes into secondary liquefaction 1.5 gms of hydrolyzate wasadded to a tared one liter volumetric containing 500 cc of DI water and1 ml of one normal HCl and the exact wt. added was recorded. This wasrepeated at 15 minute intervals out to 90 minutes with an additionalpoint at 127 minutes. These were diluted to one liter and determined fordextrose equivalence via Neocuproine method as discribed by Dygert,Li,Florida and Thomas. Determination of reducing sugar with improvedprecision (1965). Anal. Biochem 13, 368.

The results were as follows:

Time DE 15 3.2 30 4.8 45 6.3 60 7.8 75 9.4 90 10.4 127 13.1

Example 5

Isolation of Genomic DNA from DSM 12648 and DSM 12649

The strains Bacillus sp. DSM 12649 (the AA560 α-amylase) and Bacillussp. DSM 12648 (the AA349 α-amylase) were propagated in liquid TY medium(as described in Ausubel et al. (1995)). After 16 hours incubation at37° C. and 300 rpm, the cells were harvested, and genomic DNA isolatedby the method described by Pitcher et al. (1989).

Genomic Library Construction

Genomic DNA of strain DSM 12649 was partially digested with restrictionenzyme Sau3A, and size-fractionated by electrophoresis on a 0.7% agarosegel. Fragments between 2 and 10 kb in size was isolated byelectrophoresis onto DEAE-cellulose paper (Dretzen et al. (1981).

Isolated DNA fragments were ligated to BamHI digested pSJ1678 plasmidDNA, and the ligation mixture was used to transform E. coli SJ2.

Transformation

E. coli SJ2 host cells were prepared for and transformed byelectroporation using a gene PULSE™ electroporator from BIO-RAD asdescribed by the supplier.

Identification of Positive Transformant:

A DNA library in E. coli SJ2, constructed as described above, wasscreened on LB agar plates (described in Ausbel et al. (1995))containing 0.5% AZCL-amylose (Megazyme) and 10 μg/ml Chloramphenicol andincubated overnight at 37° C. Clones expressing amylase activityappeared with blue diffusion haloes. One such clone was named LiH1274.The DNA was further characterized by DNA sequencing of part of thecloned Sau3A DNA fragment.

Example 6

Determination of the DNA Sequence of the Gene Encoding Alpha-amylasefrom Strain DSM 12648 (AA349)

The clone constituting a large chromosomal fragment containing the geneencoding the amylolytic activity inserted into plasmid pSJ1678,pLiH1247, was used as template to specifically PCR amplify internal DNAfragments of the α-amylase encoding gene by the use of degenerateprimers directed towards the conserved regions in known Bacillusalpha-amylases.

The degenerate primers were directed towards the following regions/aminoacid sequences:

For36: GITA(L/V/I)W(I/L) (SEQ ID NO: 27)

For97: VY(G/A)D(V/F/L)V(M/L/I/F)NH (SEQ ID NO: 28)

For227: DG(F/I)R(F/L/I/V)DA(A/V)KH (SEQ ID NO: 29)

Rev235: DG(F/I)R(F/L/I/V)DA(A/V)KH (SEQ ID NO: 30)

Rev328: VTFV(D/E)NHD (SEQ ID NO: 31)

Rev410: GWTREG (SEQ ID NO: 32)

The various combinations of forward (For) and reverse (Rev) primers wereused in PCR and internal DNA fragments could be amplified.

The DNA fragments were purified by QIAquick spin colums (QUIGEN) andsequenced utilizing the same degenerate primers.

From sequence the DNA sequence (SEQ ID NO: 23) of the complete codingregion encoding the mature AA349 α-amylase (SEQ ID NO: 26) wasdetermined by a standard primers-walking approach.

Example 7

Determination of the DNA Sequence of the Gene Encoding Alpha Amylasefrom Strain DSM 12649 (AA560):

A preparation of chromosomal DNA from strain DSM 12649 was utilized astemplate in a similar experiment to the one described above in Example 7in order to determine the DNA sequence of the AA560 α-amylase (SEQ IDNO: 24).

Example 8

Subcloning of the AA349 α-amylase into pTVB110.

pTVB110 is a plasmid replicating in Bacillus subtilis by the use oforigin of replication from pUB110 (Gryczan, T. J. (1978) J. Bact.134:318-329). The plasmid further encodes the cat gene, conferringresistance towards chlorampenicol, obtained from plasmid pC194(Horinouchi, S. and Weisblum, B. (1982), J. Bact. 150: 815-825). Theplasmid harbors a truncated version of the Bacillus licheniformisα-amylase gene, amyL, such that the amyL promoter, signal sequence andtranscription terminator are present, but the plasmid does not providean amy-plus phenotype (halo formation on starch containing agar).

In order to express high amount of the AA349 α-amylase the mature genewas fused precisely to the amyL signal sequence so that transcription isinitiated by the amyL promoter and translocation is directed by the amyLsignal sequence.

A PstI site is found within the mature AA349 α-amylase. Since thecloning of the gene into pTVB110 would utilize the PstI site in pTVB110,the PstI site located within the AA349 α-amylase gene was destroyedduring the cloning (by introduction of a silent mutation for amino acidAlanine 88 (GCA to GCG).

Primers 188cloningN and 188(Pst-) were used to amplify an approximately280 bp fragment by PCR on plasmid pLiH1247 using the Pwo polymeraseunder conditions recommended by the manufacturer (Boehringer Mannheim).This fragment was purified from agarose gel and used as a megaprimer (G.Sarkar and S. S. Sommer (1990) Biotechniques 8: 404-407) together withprimer 188cloningC to amplify the full length gene encoding the matureamylase in a second PCR.

The resulting approximately 1480 bp fragment was digested withrestriction endonucleases PstI and SfiI and ligated with plasmid pTVB110digested with the same enzymes.

Protease and amylase deleted Bacillus subtilis strain SHa273 (mentionedin WO 95/10603) was transformed with the ligation mixture and the DNAsequence of an amy-plus transformant was verified. This plasmid isdenoted pTVB231.

Oligonucleotides:

188(Pst-):

5′ GGC GTT AAC CGC AGC TTG TAA C (SEQ ID NO: 33)

188cloningC:

5′ CCG AGC TCG GCC GGC TGG GCC GTC GAC TTA TTT GTT TAC CCA AAT AGA AAC(SEQ ID NO: 34)

188cloningN:

5′ CAT TCT GCA GCA GCG GCG CAC CAT AAT GGT ACG AAC G (SEQ ID NO: 35)

Example 9

Subcloning of the AA560 α-amylase into pTVB110.

DNA sequencing revealed a high DNA identity between α-amylases fromstains DSM12648 (AA349) and DSM 12649 (AA560). Consequently the sameoligonucleotides and strategy was utilized for the cloning of AA560α-amylase into expression vector pTVB110 resulting in plasmid pTVB232,which was then fermented using standard techniques.

Example 10

Purification of the AA560 α-amylase

The culture broth was flocculated by adding 0.01 ml 50% (w/w) CaCl₂,2H₂O, 0.0125 ml 129 (w/w) Sodium aluminate, 0.025 ml 20% C521 and 0.075ml 0.1% A130 pr. ml culture broth. A clear solution was obtained aftercentrifugation. The enzyme solution was added ammonium sulphate to afinal concentration of 1.2 M and applied on a Butyl Toyo Pearl column(100 ml) previously equilibrated in 1.2 M ammonium sulphate, 10 mMTris-HCl, pH 7.0. The amylase was eluted using 5 mM Tris-HCl, pH 7.0 andthe eluted pool was dialysed against 5 mM Tris-HCl over night. Thefraction was then subjected to ion exchange chromatography using aQ-Sepharose column (200 ml) previously equilibrated in 20 mM Tris-HCl,pH 9.0. Unbound material was washed out with the equilibration buffer,and the amylase was eluted using a linear gradient 0-1 M NaCl, 20 mMTris-HCl, pH 9.0. Purity of the amylase preparation was above 95% judgedby SDS-PAGE.

Example 11

Characterization of the AA560 α-amylase

The α-amylase activity was measured using both the Phadebas assay (37°C., pH 7.3) and the Alternative pNPG7 Assay (25° C., pH 7.1) describedabove. pH- and temperature profiles were made at selected pH- andtemperature values. The pH-profile was measured at 37° C. and thetemperature profile was measured at pH 9.0

Isoelectric Point was determined using isoelectric focusing (Pharmacia,Ampholine, pH 3.5-9.3).

TABLE 1 Specific activity and pI. Specific activity NU/ml NU/ml EnzymePhadebas pNPG7 pI AA560 (SEQ ID NO: 4) 35000 6000 7-8 SP722 (SEQ ID NO:2) 35000 6000 7-9 SP690 (SEQ ID NO: 1) 35000 7000 5-6

The result of the pH-optimum determination and temperature optimumdetermination is shown in FIG. 2 and FIG. 3, respectively.

Example 12

Washing Test

Washing performance was evaluated by washing soiled test swatches for 15and 30 mminutes at 25° C. and 40° C., respectively, in detergentsolutions with the AA560 α-amylase of the invention.

The detergents used are disclosed in Table 2 below. The A/P ModelDetergent is described in the Materials section above. The otherdetergents are commercially available detergents. Commercial detergentscontaining amylase were inactivated by microwaves before wash.

The purified recombinant AA560 α-amylase of Example 6 was added to thedetergent solutions at the concentration indicated below. The testswatches were soiled with orange rice starch (CS-28 swatches availablefrom CFT, Center for Test Material, Holland).

After washing, the swatches were evaluated by measuring the remission at460 nm using a Elrepho Remission Spectrophotometer. The results areexpressed as ΔR=remission of the swatch washed with the α-amylase minusthe remission of a swatch washed at the same conditions without theα-amylase.

TABLE 2 Detergents and wash conditions Det. Enzyme Water Dose Inactidose Temp. Time hardness Ca: Area Detergent g/l vation mg/l ° C. min pH°dH Mg A/P Model 3 − 1 25 15 10.5 6 2:1 detergent 97 Latin Omo Multi 3 −1 25 15 10.6 6 2:1 America Acao Europe Omo conc. 4 + 0.2 40 30 10.2 154:1 Powder Europe Ariel 5 + 0.2 40 30 9.0 15 4:1 Futur liquid

The results are shown in FIGS. 4-7. The results demonstrate that theα-amylase of the invention is effective in both detergents at highlyalkaline pH.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

REFERENCES CITED

Klein, C., et al., Biochemistry 1992, 31, 8740-8746.

Mizuno, H., et al., J. Mol. Biol. (1993) 234, 1282-1283.

Chang, C., et al, J. Mol. Biol. (1993) 229, 235-238.

Larson, S. B., J. Mol. Biol. (1994) 235, 1560-1584.

Lawson, C. L., J. Mol. Biol. (1994) 236, 590-600.

Qian, M., et al., J. Mol. Biol. (1993) 231, 785-799.

Brady, R. L., et al., Acta Crystallogr. sect. B, 47, 527-535.

Swift, H. J., et al., Acta Crystallogr. sect. B, 47, 535-544.

A. Kadziola, Ph.D. Thesis: “An alpha-amylase from Barley and its Complexwith a Substrate Analogue Inhibitor Studied by X-ray Crystallography”,Department of Chemistry University of Copenhagen 1993.

MacGregor, E. A., Food Hydrocolloids, 1987, Vol. 1, No. 5-6.

B. Diderichsen and L. Christiansen, Cloning of a maltogenic α-amylasefrom Bacillus stearothermophilus, FEMS Microbiol. letters: 56: pp. 53-60(1988).

Hudson et al., Practical Immunology, Third edition (1989), BlackwellScientific Publications.

Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., ColdSpring Harbor, 1989.

S. L. Beaucage and M. H. Caruthers, Tetrahedron Letters 22, 1981, pp.1859-1869

Matthes et al., The EMBO J. 3, 1984, pp. 801-805.

R. K. Saiki et al., Science 239, 1988, pp. 487-491.

Morinaga et al., (1984, Biotechnology 2:646-639)

Nelson and Long, Analytical Biochemistry 180, 1989, pp. 147-151

Hunkapiller et al., 1984, Nature 310:105-111

R. Higuchi, B. Krummel, and R. K. Saiki (1988). A general method of invitro preparation and specific mutagenesis of DNA fragments: study ofprotein and DNA interactions. Nucl. Acids Res. 16:7351-7367.

Dubnau et al., 1971, J. Mol. Biol. 56, pp. 209-221.

Gryczan et al., 1978, J. Bacteriol. 134, pp. 318-329.

S. D. Erlich, 1977, Proc. Natl. Acad. Sci. 74, pp. 1680-1682.

Boel et al., 1990, Biochemistry 29, pp. 6244-6249.

Ausubel, F. M. et al. (eds.); Current protocols in Molecular Biology;1995; John Wiley and Sons.

Harwood C. R., and Cutting S. M. (eds.); Molecular Biological Methodsfor Bacillus; 1990; John Wiley and Sons.

Diderichsen B., Wedsted U., Hedegaard L., Jensen B. R., Sjholm C.;Cloning of aldB, which encodes alpha-acetolactate decarboxylase, anexoenzyme from Bacillus brevis; J. Bacteriol., 1990, vol. 172, pp.4315-4321.

Pitcher D. G., Saunders N. A., Owen R. J.; Rapid extraction of bacterialgenomic DNA with guanidium thiocyanate; Lett. Appl. Microbiol.; 1989;vol. 8; pp. 151-156.

Dretzen G., Bellard M., Sassone-Corsi P., Chambon P.; A reliable methodfor the recovery of DNA fragments from agarose and acrylamide gels;Anal. Biochem.; 1981; vol. 112; pp. 295-298.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 35 <210> SEQ ID NO 1 <211>LENGTH: 485 <212> TYPE: PRT <213> ORGANISM: Bacillus sp. <400> SEQUENCE:1 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 1015 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 2530 Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 4045 Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 5560 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 7075 80 Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 8590 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp100 105 110 Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn ArgAsn 115 120 125 Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr LysPhe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys TrpArg Trp Tyr 145 150 155 160 His Phe Asp Gly Thr Asp Trp Asp Gln Ser ArgGln Leu Gln Asn Lys 165 170 175 Ile Tyr Lys Phe Arg Gly Thr Gly Lys AlaTrp Asp Trp Glu Val Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr LeuMet Tyr Ala Asp Val Asp Met 195 200 205 Asp His Pro Glu Val Ile His GluLeu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Asn Leu AspGly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser PheThr Arg Asp Trp Leu Thr His Val Arg Asn Thr 245 250 255 Thr Gly Lys ProMet Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala IleGlu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val 275 280 285 Phe AspVal Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 GlyTyr Tyr Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys 305 310 315320 His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325330 335 Gly Glu Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala340 345 350 Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val PheTyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala MetLys Ser 370 375 380 Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe AlaTyr Gly Thr 385 390 395 400 Gln His Asp Tyr Phe Asp His His Asp Ile IleGly Trp Thr Arg Glu 405 410 415 Gly Asn Ser Ser His Pro Asn Ser Gly LeuAla Thr Ile Met Ser Asp 420 425 430 Gly Pro Gly Gly Asn Lys Trp Met TyrVal Gly Lys Asn Lys Ala Gly 435 440 445 Gln Val Trp Arg Asp Ile Thr GlyAsn Arg Thr Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly AsnPhe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 Val Trp Val Lys Gln485 <210> SEQ ID NO 2 <211> LENGTH: 485 <212> TYPE: PRT <213> ORGANISM:Bacillus sp. <400> SEQUENCE: 2 His His Asn Gly Thr Asn Gly Thr Met MetGln Tyr Phe Glu Trp His 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp AsnArg Leu Arg Asp Asp Ala Ser 20 25 30 Asn Leu Arg Asn Arg Gly Ile Thr AlaIle Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Thr Ser Gln Asn Asp Val GlyTyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys GlyThr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Ser Gln Leu Glu Ser AlaIle His Ala Leu Lys Asn Asn Gly 85 90 95 Val Gln Val Tyr Gly Asp Val ValMet Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Asn Val Leu AlaVal Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Ile Ser Gly AspTyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg GlyAsn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe AspGly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg 165 170 175 Ile TyrLys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190 SerGlu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr 210 215220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg AsnAla 245 250 255 Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys AsnAsp Leu 260 265 270 Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp AsnHis Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn AlaSer Asn Ser Gly 290 295 300 Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn GlyThr Val Val Gln Lys 305 310 315 320 His Pro Met His Ala Val Thr Phe ValAsp Asn His Asp Ser Gln Pro 325 330 335 Gly Glu Ser Leu Glu Ser Phe ValGln Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Ile Leu Thr ArgGlu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly IlePro Thr His Ser Val Pro Ala Met Lys Ala 370 375 380 Lys Ile Asp Pro IleLeu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr 385 390 395 400 Gln His AspTyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly AsnThr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 GlyPro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly 435 440 445Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile 450 455460 Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465470 475 480 Ile Trp Val Lys Arg 485 <210> SEQ ID NO 3 <211> LENGTH: 514<212> TYPE: PRT <213> ORGANISM: B. stearothermophilus <400> SEQUENCE: 3Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu 1 5 1015 Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 2530 Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 4045 Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 50 5560 Leu Gly Glu Phe Asn Gln Lys Gly Ala Val Arg Thr Lys Tyr Gly Thr 65 7075 80 Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met 8590 95 Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly100 105 110 Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg AsnGln 115 120 125 Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys PheAsp Phe 130 135 140 Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp ArgTrp Tyr His 145 150 155 160 Phe Asp Gly Val Asp Trp Asp Glu Ser Arg LysLeu Ser Arg Ile Tyr 165 170 175 Lys Phe Arg Gly Ile Gly Lys Ala Trp AspTrp Glu Val Asp Thr Glu 180 185 190 Asn Gly Asn Tyr Asp Tyr Leu Met TyrAla Asp Leu Asp Met Asp His 195 200 205 Pro Glu Val Val Thr Glu Leu LysSer Trp Gly Lys Trp Tyr Val Asn 210 215 220 Thr Thr Asn Ile Asp Gly PheArg Leu Asp Ala Val Lys His Ile Lys 225 230 235 240 Phe Ser Phe Phe ProAsp Trp Leu Ser Asp Val Arg Ser Gln Thr Gly 245 250 255 Lys Pro Leu PheThr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys 260 265 270 Leu His AsnTyr Ile Met Lys Thr Asn Gly Thr Met Ser Leu Phe Asp 275 280 285 Ala ProLeu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Thr 290 295 300 PheAsp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro 305 310 315320 Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln 325330 335 Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala340 345 350 Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr GlyAsp 355 360 365 Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys SerLys Ile 370 375 380 Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr GlyThr Gln His 385 390 395 400 Asp Tyr Leu Asp His Ser Asp Ile Ile Gly TrpThr Arg Glu Gly Val 405 410 415 Thr Glu Lys Pro Gly Ser Gly Leu Ala AlaLeu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ser Lys Trp Met Tyr Val GlyLys Gln His Ala Gly Lys Val 435 440 445 Phe Tyr Asp Leu Thr Gly Asn ArgSer Asp Thr Val Thr Ile Asn Ser 450 455 460 Asp Gly Trp Gly Glu Phe LysVal Asn Gly Gly Ser Val Ser Val Trp 465 470 475 480 Val Pro Arg Lys ThrThr Val Ser Thr Ile Ala Trp Ser Ile Thr Thr 485 490 495 Arg Pro Trp ThrAsp Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val 500 505 510 Ala Trp<210> SEQ ID NO 4 <211> LENGTH: 483 <212> TYPE: PRT <213> ORGANISM: B.licheniformis <400> SEQUENCE: 4 Ala Asn Leu Asn Gly Thr Leu Met Gln TyrPhe Glu Trp Tyr Met Pro 1 5 10 15 Asn Asp Gly Gln His Trp Arg Arg LeuGln Asn Asp Ser Ala Tyr Leu 20 25 30 Ala Glu His Gly Ile Thr Ala Val TrpIle Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gln Ala Asp Val Gly Tyr GlyAla Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe His Gln Lys Gly Thr ValArg Thr Lys Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu Gln Ser Ala Ile LysSer Leu His Ser Arg Asp Ile Asn 85 90 95 Val Tyr Gly Asp Val Val Ile AsnHis Lys Gly Gly Ala Asp Ala Thr 100 105 110 Glu Asp Val Thr Ala Val GluVal Asp Pro Ala Asp Arg Asn Arg Val 115 120 125 Ile Ser Gly Glu His LeuIle Lys Ala Trp Thr His Phe His Phe Pro 130 135 140 Gly Arg Gly Ser ThrTyr Ser Asp Phe Lys Trp His Trp Tyr His Phe 145 150 155 160 Asp Gly ThrAsp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170 175 Phe GlnGly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190 TyrAsp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195 200 205Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln 210 215220 Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe 225230 235 240 Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys GluMet 245 250 255 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala LeuGlu Asn 260 265 270 Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe AspVal Pro Leu 275 280 285 His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly GlyGly Tyr Asp Met 290 295 300 Arg Lys Leu Leu Asn Gly Thr Val Val Ser LysHis Pro Leu Lys Ser 305 310 315 320 Val Thr Phe Val Asp Asn His Asp ThrGln Pro Gly Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Thr Trp Phe LysPro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser Gly Tyr ProGln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Asp Ser GlnArg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380 Glu Pro Ile Leu LysAla Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400 Asp Tyr PheAsp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser SerVal Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 GlyGly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435 440 445Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser 450 455460 Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr 465470 475 480 Val Gln Arg <210> SEQ ID NO 5 <211> LENGTH: 480 <212> TYPE:PRT <213> ORGANISM: B. amyloliquefaciens <400> SEQUENCE: 5 Val Asn GlyThr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp 1 5 10 15 Gly GlnHis Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp 20 25 30 Ile GlyIle Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser 35 40 45 Gln SerAsp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu 50 55 60 Phe GlnGln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu 65 70 75 80 LeuGln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr 85 90 95 GlyAsp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp 100 105 110Val Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser 115 120125 Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg 130135 140 Gly Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly145 150 155 160 Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe LysPhe Arg 165 170 175 Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser GluAsn Gly Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr AspHis Pro Asp Val 195 200 205 Val Ala Glu Thr Lys Lys Trp Gly Ile Trp TyrAla Asn Glu Leu Ser 210 215 220 Leu Asp Gly Phe Arg Ile Asp Ala Ala LysHis Ile Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val Gln Ala ValArg Gln Ala Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala Glu Tyr TrpGln Asn Asn Ala Gly Lys Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr SerPhe Asn Gln Ser Val Phe Asp Val Pro Leu 275 280 285 His Phe Asn Leu GlnAla Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met 290 295 300 Arg Arg Leu LeuAsp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala 305 310 315 320 Val ThrPhe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335 SerThr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360365 Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile 370375 380 Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His385 390 395 400 Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg GluGly Asp 405 410 415 Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile ThrAsp Gly Pro 420 425 430 Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys AsnAla Gly Glu Thr 435 440 445 Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp ThrVal Lys Ile Gly Ser 450 455 460 Asp Gly Trp Gly Glu Phe His Val Asn AspGly Ser Val Ser Ile Tyr 465 470 475 480 <210> SEQ ID NO 6 <211> LENGTH:485 <212> TYPE: PRT <213> ORGANISM: Bacillus sp. <400> SEQUENCE: 6 HisHis Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn Ser Asp Ala Ser 20 25 30Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 7580 Thr Arg Ser Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 9095 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100105 110 Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn115 120 125 Gln Glu Val Thr Gly Glu Tyr Thr Ile Glu Ala Trp Thr Arg PheAsp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser Ser Phe Lys Trp ArgTrp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg ArgLeu Asn Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly His Gly Lys Ala TrpAsp Trp Glu Val Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu MetTyr Ala Asp Ile Asp Met 195 200 205 Asp His Pro Glu Val Val Asn Glu LeuArg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Gly Leu Asp GlyPhe Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe ThrArg Asp Trp Ile Asn His Val Arg Ser Ala 245 250 255 Thr Gly Lys Asn MetPhe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile GluAsn Tyr Leu Gln Lys Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp ValPro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly 290 295 300 Gly AsnTyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln Arg 305 310 315 320His Pro Ser His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330335 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340345 350 Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met ArgSer 370 375 380 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala TyrGly Lys 385 390 395 400 Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile GlyTrp Thr Arg Glu 405 410 415 Gly Asn Thr Ala His Pro Asn Ser Gly Leu AlaThr Ile Met Ser Asp 420 425 430 Gly Ala Gly Gly Ser Lys Trp Met Phe ValGly Arg Asn Lys Ala Gly 435 440 445 Gln Val Trp Ser Asp Ile Thr Gly AsnArg Thr Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn PheSer Val Asn Gly Gly Ser Val Ser 465 470 475 480 Ile Trp Val Asn Lys 485<210> SEQ ID NO 7 <211> LENGTH: 485 <212> TYPE: PRT <213> ORGANISM:Bacillus sp. <400> SEQUENCE: 7 His His Asn Gly Thr Asn Gly Thr Met MetGln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp AsnArg Leu Arg Asp Asp Ala Ala 20 25 30 Asn Leu Lys Ser Lys Gly Ile Thr AlaVal Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Thr Ser Gln Asn Asp Val GlyTyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys GlyThr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Asn Gln Leu Gln Ala AlaVal Thr Ser Leu Lys Asn Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val ValMet Asn His Lys Gly Gly Ala Asp 100 105 110 Gly Thr Glu Ile Val Asn AlaVal Glu Val Asn Arg Ser Asn Arg Asn 115 120 125 Gln Glu Thr Ser Gly GluTyr Ala Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg GlyAsn Asn His Ser Ser Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe AspGly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys 165 170 175 Ile TyrLys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190 ThrGlu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205Asp His Pro Glu Val Ile His Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg AsnThr 245 250 255 Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys AsnAsp Leu 260 265 270 Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp AsnHis Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn AlaSer Asn Ser Gly 290 295 300 Gly Tyr Tyr Asp Met Arg Asn Ile Leu Asn GlySer Val Val Gln Lys 305 310 315 320 His Pro Thr His Ala Val Thr Phe ValAsp Asn His Asp Ser Gln Pro 325 330 335 Gly Glu Ala Leu Glu Ser Phe ValGln Gln Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Val Leu Thr ArgGlu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly IlePro Thr His Gly Val Pro Ala Met Lys Ser 370 375 380 Lys Ile Asp Pro LeuLeu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr 385 390 395 400 Gln His AspTyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly AsnSer Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 GlyPro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly 435 440 445Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465470 475 480 Val Trp Val Lys Gln 485 <210> SEQ ID NO 8 <211> LENGTH: 485<212> TYPE: PRT <213> ORGANISM: Bacillus sp. <400> SEQUENCE: 8 His HisAsn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His 1 5 10 15 LeuPro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30 AsnLeu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45 LysGly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 AspLeu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly 85 90 95Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105110 Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115120 125 Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp130 135 140 Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg TrpTyr 145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln PheGln Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp AspTrp Glu Val Asp 180 185 190 Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met TyrAla Asp Val Asp Met 195 200 205 Asp His Pro Glu Val Val Asn Glu Leu ArgArg Trp Gly Glu Trp Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly PheArg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr ArgAsp Trp Leu Thr His Val Arg Asn Ala 245 250 255 Thr Gly Lys Glu Met PheAla Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Leu Glu AsnTyr Leu Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp Val ProLeu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Asn TyrAsp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys 305 310 315 320 HisPro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala 340 345350 Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala370 375 380 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr GlyThr 385 390 395 400 Gln His Asp Tyr Phe Asp His His Asn Ile Ile Gly TrpThr Arg Glu 405 410 415 Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala ThrIle Met Ser Asp 420 425 430 Gly Pro Gly Gly Glu Lys Trp Met Tyr Val GlyGln Asn Lys Ala Gly 435 440 445 Gln Val Trp His Asp Ile Thr Gly Asn LysPro Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Ala Asn Phe SerVal Asn Gly Gly Ser Val Ser 465 470 475 480 Ile Trp Val Lys Arg 485<210> SEQ ID NO 9 <211> LENGTH: 1455 <212> TYPE: DNA <213> ORGANISM:Bacillus sp. <400> SEQUENCE: 9 catcataatg gaacaaatgg tactatgatgcaatatttcg aatggtattt gccaaatgac 60 gggaatcatt ggaacaggtt gagggatgacgcagctaact taaagagtaa agggataaca 120 gctgtatgga tcccacctgc atggaaggggacttcccaga atgatgtagg ttatggagcc 180 tatgatttat atgatcttgg agagtttaaccagaagggga cggttcgtac aaaatatgga 240 acacgcaacc agctacaggc tgcggtgacctctttaaaaa ataacggcat tcaggtatat 300 ggtgatgtcg tcatgaatca taaaggtggagcagatggta cggaaattgt aaatgcggta 360 gaagtgaatc ggagcaaccg aaaccaggaaacctcaggag agtatgcaat agaagcgtgg 420 acaaagtttg attttcctgg aagaggaaataaccattcca gctttaagtg gcgctggtat 480 cattttgatg ggacagattg ggatcagtcacgccagcttc aaaacaaaat atataaattc 540 aggggaacag gcaaggcctg ggactgggaagtcgatacag agaatggcaa ctatgactat 600 cttatgtatg cagacgtgga tatggatcacccagaagtaa tacatgaact tagaaactgg 660 ggagtgtggt atacgaatac actgaaccttgatggattta gaatagatgc agtgaaacat 720 ataaaatata gctttacgag agattggcttacacatgtgc gtaacaccac aggtaaacca 780 atgtttgcag tggctgagtt ttggaaaaatgaccttggtg caattgaaaa ctatttgaat 840 aaaacaagtt ggaatcactc ggtgtttgatgttcctctcc actataattt gtacaatgca 900 tctaatagcg gtggttatta tgatatgagaaatattttaa atggttctgt ggtgcaaaaa 960 catccaacac atgccgttac ttttgttgataaccatgatt ctcagcccgg ggaagcattg 1020 gaatcctttg ttcaacaatg gtttaaaccacttgcatatg cattggttct gacaagggaa 1080 caaggttatc cttccgtatt ttatggggattactacggta tcccaaccca tggtgttccg 1140 gctatgaaat ctaaaataga ccctcttctgcaggcacgtc aaacttttgc ctatggtacg 1200 cagcatgatt actttgatca tcatgatattatcggttgga caagagaggg aaatagctcc 1260 catccaaatt caggccttgc caccattatgtcagatggtc caggtggtaa caaatggatg 1320 tatgtgggga aaaataaagc gggacaagtttggagagata ttaccggaaa taggacaggc 1380 accgtcacaa ttaatgcaga cggatggggtaatttctctg ttaatggagg gtccgtttcg 1440 gtttgggtga agcaa 1455 <210> SEQ IDNO 10 <211> LENGTH: 1455 <212> TYPE: DNA <213> ORGANISM: Bacillus sp.<400> SEQUENCE: 10 catcataatg ggacaaatgg gacgatgatg caatactttgaatggcactt gcctaatgat 60 gggaatcact ggaatagatt aagagatgat gctagtaatctaagaaatag aggtataacc 120 gctatttgga ttccgcctgc ctggaaaggg acttcgcaaaatgatgtggg gtatggagcc 180 tatgatcttt atgatttagg ggaatttaat caaaaggggacggttcgtac taagtatggg 240 acacgtagtc aattggagtc tgccatccat gctttaaagaataatggcgt tcaagtttat 300 ggggatgtag tgatgaacca taaaggagga gctgatgctacagaaaacgt tcttgctgtc 360 gaggtgaatc caaataaccg gaatcaagaa atatctggggactacacaat tgaggcttgg 420 actaagtttg attttccagg gaggggtaat acatactcagactttaaatg gcgttggtat 480 catttcgatg gtgtagattg ggatcaatca cgacaattccaaaatcgtat ctacaaattc 540 cgaggtgatg gtaaggcatg ggattgggaa gtagattcggaaaatggaaa ttatgattat 600 ttaatgtatg cagatgtaga tatggatcat ccggaggtagtaaatgagct tagaagatgg 660 ggagaatggt atacaaatac attaaatctt gatggatttaggatcgatgc ggtgaagcat 720 attaaatata gctttacacg tgattggttg acccatgtaagaaacgcaac gggaaaagaa 780 atgtttgctg ttgctgaatt ttggaaaaat gatttaggtgccttggagaa ctatttaaat 840 aaaacaaact ggaatcattc tgtctttgat gtcccccttcattataatct ttataacgcg 900 tcaaatagtg gaggcaacta tgacatggca aaacttcttaatggaacggt tgttcaaaag 960 catccaatgc atgccgtaac ttttgtggat aatcacgattctcaacctgg ggaatcatta 1020 gaatcatttg tacaagaatg gtttaagcca cttgcttatgcgcttatttt aacaagagaa 1080 caaggctatc cctctgtctt ctatggtgac tactatggaattccaacaca tagtgtccca 1140 gcaatgaaag ccaagattga tccaatctta gaggcgcgtcaaaattttgc atatggaaca 1200 caacatgatt attttgacca tcataatata atcggatggacacgtgaagg aaataccacg 1260 catcccaatt caggacttgc gactatcatg tcggatgggccagggggaga gaaatggatg 1320 tacgtagggc aaaataaagc aggtcaagtt tggcatgacataactggaaa taaaccagga 1380 acagttacga tcaatgcaga tggatgggct aatttttcagtaaatggagg atctgtttcc 1440 atttgggtga aacga 1455 <210> SEQ ID NO 11<211> LENGTH: 1548 <212> TYPE: DNA <213> ORGANISM: B. stearothermophilus<400> SEQUENCE: 11 gccgcaccgt ttaacggcac catgatgcag tattttgaatggtacttgcc ggatgatggc 60 acgttatgga ccaaagtggc caatgaagcc aacaacttatccagccttgg catcaccgct 120 ctttggctgc cgcccgctta caaaggaaca agccgcagcgacgtagggta cggagtatac 180 gacttgtatg acctcggcga attcaatcaa aaagggaccgtccgcacaaa atacggaaca 240 aaagctcaat atcttcaagc cattcaagcc gcccacgccgctggaatgca agtgtacgcc 300 gatgtcgtgt tcgaccataa aggcggcgct gacggcacggaatgggtgga cgccgtcgaa 360 gtcaatccgt ccgaccgcaa ccaagaaatc tcgggcacctatcaaatcca agcatggacg 420 aaatttgatt ttcccgggcg gggcaacacc tactccagctttaagtggcg ctggtaccat 480 tttgacggcg ttgattggga cgaaagccga aaattgagccgcatttacaa attccgcggc 540 atcggcaaag cgtgggattg ggaagtagac acggaaaacggaaactatga ctacttaatg 600 tatgccgacc ttgatatgga tcatcccgaa gtcgtgaccgagctgaaaaa ctgggggaaa 660 tggtatgtca acacaacgaa cattgatggg ttccggcttgatgccgtcaa gcatattaag 720 ttcagttttt ttcctgattg gttgtcgtat gtgcgttctcagactggcaa gccgctattt 780 accgtcgggg aatattggag ctatgacatc aacaagttgcacaattacat tacgaaaaca 840 gacggaacga tgtctttgtt tgatgccccg ttacacaacaaattttatac cgcttccaaa 900 tcagggggcg catttgatat gcgcacgtta atgaccaatactctcatgaa agatcaaccg 960 acattggccg tcaccttcgt tgataatcat gacaccgaacccggccaagc gctgcagtca 1020 tgggtcgacc catggttcaa accgttggct tacgcctttattctaactcg gcaggaagga 1080 tacccgtgcg tcttttatgg tgactattat ggcattccacaatataacat tccttcgctg 1140 aaaagcaaaa tcgatccgct cctcatcgcg cgcagggattatgcttacgg aacgcaacat 1200 gattatcttg atcactccga catcatcggg tggacaagggaagggggcac tgaaaaacca 1260 ggatccggac tggccgcact gatcaccgat gggccgggaggaagcaaatg gatgtacgtt 1320 ggcaaacaac acgctggaaa agtgttctat gaccttaccggcaaccggag tgacaccgtc 1380 accatcaaca gtgatggatg gggggaattc aaagtcaatggcggttcggt ttcggtttgg 1440 gttcctagaa aaacgaccgt ttctaccatc gctcggccgatcacaacccg accgtggact 1500 ggtgaattcg tccgttggac cgaaccacgg ttggtggcatggccttga 1548 <210> SEQ ID NO 12 <211> LENGTH: 1920 <212> TYPE: DNA<213> ORGANISM: B. licheniformis <400> SEQUENCE: 12 cggaagattggaagtacaaa aataagcaaa agattgtcaa tcatgtcatg agccatgcgg 60 gagacggaaaaatcgtctta atgcacgata tttatgcaac gttcgcagat gctgctgaag 120 agattattaaaaagctgaaa gcaaaaggct atcaattggt aactgtatct cagcttgaag 180 aagtgaagaagcagagaggc tattgaataa atgagtagaa gcgccatatc ggcgcttttc 240 ttttggaagaaaatataggg aaaatggtac ttgttaaaaa ttcggaatat ttatacaaca 300 tcatatgtttcacattgaaa ggggaggaga atcatgaaac aacaaaaacg gctttacgcc 360 cgattgctgacgctgttatt tgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420 gcaaatcttaatgggacgct gatgcagtat tttgaatggt acatgcccaa tgacggccaa 480 cattggaggcgtttgcaaaa cgactcggca tatttggctg aacacggtat tactgccgtc 540 tggattcccccggcatataa gggaacgagc caagcggatg tgggctacgg tgcttacgac 600 ctttatgatttaggggagtt tcatcaaaaa gggacggttc ggacaaagta cggcacaaaa 660 ggagagctgcaatctgcgat caaaagtctt cattcccgcg acattaacgt ttacggggat 720 gtggtcatcaaccacaaagg cggcgctgat gcgaccgaag atgtaaccgc ggttgaagtc 780 gatcccgctgaccgcaaccg cgtaatttca ggagaacacc taattaaagc ctggacacat 840 tttcattttccggggcgcgg cagcacatac agcgatttta aatggcattg gtaccatttt 900 gacggaaccgattgggacga gtcccgaaag ctgaaccgca tctataagtt tcaaggaaag 960 gcttgggattgggaagtttc caatgaaaac ggcaactatg attatttgat gtatgccgac 1020 atcgattatgaccatcctga tgtcgcagca gaaattaaga gatggggcac ttggtatgcc 1080 aatgaactgcaattggacgg tttccgtctt gatgctgtca aacacattaa attttctttt 1140 ttgcgggattgggttaatca tgtcagggaa aaaacgggga aggaaatgtt tacggtagct 1200 gaatattggcagaatgactt gggcgcgctg gaaaactatt tgaacaaaac aaattttaat 1260 cattcagtgtttgacgtgcc gcttcattat cagttccatg ctgcatcgac acagggaggc 1320 ggctatgatatgaggaaatt gctgaacggt acggtcgttt ccaagcatcc gttgaaatcg 1380 gttacatttgtcgataacca tgatacacag ccggggcaat cgcttgagtc gactgtccaa 1440 acatggtttaagccgcttgc ttacgctttt attctcacaa gggaatctgg ataccctcag 1500 gttttctacggggatatgta cgggacgaaa ggagactccc agcgcgaaat tcctgccttg 1560 aaacacaaaattgaaccgat cttaaaagcg agaaaacagt atgcgtacgg agcacagcat 1620 gattatttcgaccaccatga cattgtcggc tggacaaggg aaggcgacag ctcggttgca 1680 aattcaggtttggcggcatt aataacagac ggacccggtg gggcaaagcg aatgtatgtc 1740 ggccggcaaaacgccggtga gacatggcat gacattaccg gaaaccgttc ggagccggtt 1800 gtcatcaattcggaaggctg gggagagttt cacgtaaacg gcgggtcggt ttcaatttat 1860 gttcaaagatagaagagcag agaggacgga tttcctgaag gaaatccgtt tttttatttt 1920 <210> SEQ IDNO 13 <211> LENGTH: 2084 <212> TYPE: DNA <213> ORGANISM: B.amyloliquefaciens <400> SEQUENCE: 13 gccccgcaca tacgaaaaga ctggctgaaaacattgagcc tttgatgact gatgatttgg 60 ctgaagaagt ggatcgattg tttgagaaaagaagaagacc ataaaaatac cttgtctgtc 120 atcagacagg gtatttttta tgctgtccagactgtccgct gtgtaaaaat aaggaataaa 180 ggggggttgt tattatttta ctgatatgtaaaatataatt tgtataagaa aatgagaggg 240 agaggaaaca tgattcaaaa acgaaagcggacagtttcgt tcagacttgt gcttatgtgc 300 acgctgttat ttgtcagttt gccgattacaaaaacatcag ccgtaaatgg cacgctgatg 360 cagtattttg aatggtatac gccgaacgacggccagcatt ggaaacgatt gcagaatgat 420 gcggaacatt tatcggatat cggaatcactgccgtctgga ttcctcccgc atacaaagga 480 ttgagccaat ccgataacgg atacggaccttatgatttgt atgatttagg agaattccag 540 caaaaaggga cggtcagaac gaaatacggcacaaaatcag agcttcaaga tgcgatcggc 600 tcactgcatt cccggaacgt ccaagtatacggagatgtgg ttttgaatca taaggctggt 660 gctgatgcaa cagaagatgt aactgccgtcgaagtcaatc cggccaatag aaatcaggaa 720 acttcggagg aatatcaaat caaagcgtggacggattttc gttttccggg ccgtggaaac 780 acgtacagtg attttaaatg gcattggtatcatttcgacg gagcggactg ggatgaatcc 840 cggaagatca gccgcatctt taagtttcgtggggaaggaa aagcgtggga ttgggaagta 900 tcaagtgaaa acggcaacta tgactatttaatgtatgctg atgttgacta cgaccaccct 960 gatgtcgtgg cagagacaaa aaaatggggtatctggtatg cgaatgaact gtcattagac 1020 ggcttccgta ttgatgccgc caaacatattaaattttcat ttctgcgtga ttgggttcag 1080 gcggtcagac aggcgacggg aaaagaaatgtttacggttg cggagtattg gcagaataat 1140 gccgggaaac tcgaaaacta cttgaataaaacaagcttta atcaatccgt gtttgatgtt 1200 ccgcttcatt tcaatttaca ggcggcttcctcacaaggag gcggatatga tatgaggcgt 1260 ttgctggacg gtaccgttgt gtccaggcatccggaaaagg cggttacatt tgttgaaaat 1320 catgacacac agccgggaca gtcattggaatcgacagtcc aaacttggtt taaaccgctt 1380 gcatacgcct ttattttgac aagagaatccggttatcctc aggtgttcta tggggatatg 1440 tacgggacaa aagggacatc gccaaaggaaattccctcac tgaaagataa tatagagccg 1500 attttaaaag cgcgtaagga gtacgcatacgggccccagc acgattatat tgaccacccg 1560 gatgtgatcg gatggacgag ggaaggtgacagctccgccg ccaaatcagg tttggccgct 1620 ttaatcacgg acggacccgg cggatcaaagcggatgtatg ccggcctgaa aaatgccggc 1680 gagacatggt atgacataac gggcaaccgttcagatactg taaaaatcgg atctgacggc 1740 tggggagagt ttcatgtaaa cgatgggtccgtctccattt atgttcagaa ataaggtaat 1800 aaaaaaacac ctccaagctg agtgcgggtatcagcttgga ggtgcgttta ttttttcagc 1860 cgtatgacaa ggtcggcatc aggtgtgacaaatacggtat gctggctgtc ataggtgaca 1920 aatccgggtt ttgcgccgtt tggctttttcacatgtctga tttttgtata atcaacaggc 1980 acggagccgg aatctttcgc cttggaaaaataagcggcga tcgtagctgc ttccaatatg 2040 gattgttcat cgggatcgct gcttttaatcacaacgtggg atcc 2084 <210> SEQ ID NO 14 <211> LENGTH: 1455 <212> TYPE:DNA <213> ORGANISM: Bacillus sp. <400> SEQUENCE: 14 catcataatggaacaaatgg tactatgatg caatatttcg aatggtattt gccaaatgac 60 gggaatcattggaacaggtt gagggatgac gcagctaact taaagagtaa agggataaca 120 gctgtatggatcccacctgc atggaagggg acttcccaga atgatgtagg ttatggagcc 180 tatgatttatatgatcttgg agagtttaac cagaagggga cggttcgtac aaaatatgga 240 acacgcaaccagctacaggc tgcggtgacc tctttaaaaa ataacggcat tcaggtatat 300 ggtgatgtcgtcatgaatca taaaggtgga gcagatggta cggaaattgt aaatgcggta 360 gaagtgaatcggagcaaccg aaaccaggaa acctcaggag agtatgcaat agaagcgtgg 420 acaaagtttgattttcctgg aagaggaaat aaccattcca gctttaagtg gcgctggtat 480 cattttgatgggacagattg ggatcagtca cgccagcttc aaaacaaaat atataaattc 540 aggggaacaggcaaggcctg ggactgggaa gtcgatacag agaatggcaa ctatgactat 600 cttatgtatgcagacgtgga tatggatcac ccagaagtaa tacatgaact tagaaactgg 660 ggagtgtggtatacgaatac actgaacctt gatggattta gaatagatgc agtgaaacat 720 ataaaatatagctttacgag agattggctt acacatgtgc gtaacaccac aggtaaacca 780 atgtttgcagtggctgagtt ttggaaaaat gaccttggtg caattgaaaa ctatttgaat 840 aaaacaagttggaatcactc ggtgtttgat gttcctctcc actataattt gtacaatgca 900 tctaatagcggtggttatta tgatatgaga aatattttaa atggttctgt ggtgcaaaaa 960 catccaacacatgccgttac ttttgttgat aaccatgatt ctcagcccgg ggaagcattg 1020 gaatcctttgttcaacaatg gtttaaacca cttgcatatg cattggttct gacaagggaa 1080 caaggttatccttccgtatt ttatggggat tactacggta tcccaaccca tggtgttccg 1140 gctatgaaatctaaaataga ccctcttctg caggcacgtc aaacttttgc ctatggtacg 1200 cagcatgattactttgatca tcatgatatt atcggttgga caagagaggg aaatagctcc 1260 catccaaattcaggccttgc caccattatg tcagatggtc caggtggtaa caaatggatg 1320 tatgtggggaaaaataaagc gggacaagtt tggagagata ttaccggaaa taggacaggc 1380 accgtcacaattaatgcaga cggatggggt aatttctctg ttaatggagg gtccgtttcg 1440 gtttgggtgaagcaa 1455 <210> SEQ ID NO 15 <211> LENGTH: 1455 <212> TYPE: DNA <213>ORGANISM: Bacillus sp. <400> SEQUENCE: 15 catcataatg ggacaaatgggacgatgatg caatactttg aatggcactt gcctaatgat 60 gggaatcact ggaatagattaagagatgat gctagtaatc taagaaatag aggtataacc 120 gctatttgga ttccgcctgcctggaaaggg acttcgcaaa atgatgtggg gtatggagcc 180 tatgatcttt atgatttaggggaatttaat caaaagggga cggttcgtac taagtatggg 240 acacgtagtc aattggagtctgccatccat gctttaaaga ataatggcgt tcaagtttat 300 ggggatgtag tgatgaaccataaaggagga gctgatgcta cagaaaacgt tcttgctgtc 360 gaggtgaatc caaataaccggaatcaagaa atatctgggg actacacaat tgaggcttgg 420 actaagtttg attttccagggaggggtaat acatactcag actttaaatg gcgttggtat 480 catttcgatg gtgtagattgggatcaatca cgacaattcc aaaatcgtat ctacaaattc 540 cgaggtgatg gtaaggcatgggattgggaa gtagattcgg aaaatggaaa ttatgattat 600 ttaatgtatg cagatgtagatatggatcat ccggaggtag taaatgagct tagaagatgg 660 ggagaatggt atacaaatacattaaatctt gatggattta ggatcgatgc ggtgaagcat 720 attaaatata gctttacacgtgattggttg acccatgtaa gaaacgcaac gggaaaagaa 780 atgtttgctg ttgctgaattttggaaaaat gatttaggtg ccttggagaa ctatttaaat 840 aaaacaaact ggaatcattctgtctttgat gtcccccttc attataatct ttataacgcg 900 tcaaatagtg gaggcaactatgacatggca aaacttctta atggaacggt tgttcaaaag 960 catccaatgc atgccgtaacttttgtggat aatcacgatt ctcaacctgg ggaatcatta 1020 gaatcatttg tacaagaatggtttaagcca cttgcttatg cgcttatttt aacaagagaa 1080 caaggctatc cctctgtcttctatggtgac tactatggaa ttccaacaca tagtgtccca 1140 gcaatgaaag ccaagattgatccaatctta gaggcgcgtc aaaattttgc atatggaaca 1200 caacatgatt attttgaccatcataatata atcggatgga cacgtgaagg aaataccacg 1260 catcccaatt caggacttgcgactatcatg tcggatgggc cagggggaga gaaatggatg 1320 tacgtagggc aaaataaagcaggtcaagtt tggcatgaca taactggaaa taaaccagga 1380 acagttacga tcaatgcagatggatgggct aatttttcag taaatggagg atctgtttcc 1440 atttgggtga aacga 1455<210> SEQ ID NO 16 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer BSG1<400> SEQUENCE: 16 ccatgatgca gtattttgaa tgg 23 <210> SEQ ID NO 17 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Primer BSG3 <400> SEQUENCE: 17gtcaccataa aagacgcacg gg 22 <210> SEQ ID NO 18 <211> LENGTH: 68 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Primer BSGM1 <400> SEQUENCE: 18 gtcatagttt ccgaattccgtgtctacttc ccaatcccaa tcccaagctt tgccgcggaa 60 tttgtaaa 68 <210> SEQ IDNO 19 <211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Primer BSGM2 <400>SEQUENCE: 19 ctacttccca atcccaagct ttgccgcgga atttgtaaat g 41 <210> SEQID NO 20 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Primer BSGM3 <400>SEQUENCE: 20 ggatgatcca tgtcaaagtc ggcatac 27 <210> SEQ ID NO 21 <211>LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Primer BSGM4 <400> SEQUENCE: 21ctcggtcacc acgtggggat gatcc 25 <210> SEQ ID NO 22 <211> LENGTH: 24 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Primer BSGM5 <400> SEQUENCE: 22 ccagtttttc agctgggtca cgac24 <210> SEQ ID NO 23 <211> LENGTH: 1458 <212> TYPE: DNA <213> ORGANISM:Bacillus sp. <220> FEATURE: <221> NAME/KEY: mat_peptide <222> LOCATION:(1)...(1458) <221> NAME/KEY: CDS <222> LOCATION: (1)...(1458) <400>SEQUENCE: 23 cac cat aat ggt acg aac ggc aca atg atg cag tac ttt gaa tggtat 48 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 15 10 15 cta cca aat gac gga aac cat tgg aat aga tta agg tct gat gca agt96 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser 20 2530 aac cta aaa gat aaa ggg atc tca gcg gtt tgg att cct cct gca tgg 144Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp 35 40 45aag ggt gcc tct caa aat gat gtg ggg tat ggt gct tat gat ctg tat 192 LysGly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 gattta gga gaa ttc aat caa aaa gga acc att cgt aca aaa tat gga 240 Asp LeuGly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly 65 70 75 80 acgcgc aat cag tta caa gct gca gtt aac gcc ttg aaa agt aat gga 288 Thr ArgAsn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 85 90 95 att caagtg tat ggc gat gtt gta atg aat cat aaa ggg gga gca gac 336 Ile Gln ValTyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 gct accgaa atg gtt agg gca gtt gaa gta aac ccg aat aat aga aat 384 Ala Thr GluMet Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 caa gaagtg tcc ggt gaa tat aca att gag gct tgg aca aag ttt gac 432 Gln Glu ValSer Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 ttt ccagga cga ggt aat act cat tca aac ttc aaa tgg aga tgg tat 480 Phe Pro GlyArg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150 155 160 cacttt gat gga gta gat tgg gat cag tca cgt aag ctg aac aat cga 528 His PheAsp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg 165 170 175 atttat aaa ttt aga ggt gat gga aaa ggg tgg gat tgg gaa gtc gat 576 Ile TyrLys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp 180 185 190 acagaa aac ggt aac tat gat tac cta atg tat gca gat att gac atg 624 Thr GluAsn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205 gatcac cca gag gta gtg aat gag cta aga aat tgg ggt gtt tgg tat 672 Asp HisPro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 acgaat aca tta ggc ctt gat ggt ttt aga ata gat gca gta aaa cat 720 Thr AsnThr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240ata aaa tac agc ttt act cgt gat tgg att aat cat gtt aga agt gca 768 IleLys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala 245 250 255act ggc aaa aat atg ttt gcg gtt gcg gaa ttt tgg aaa aat gat tta 816 ThrGly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270ggt gct att gaa aac tat tta aac aaa aca aac tgg aac cat tca gtc 864 GlyAla Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285ttt gat gtt ccg ctg cac tat aac ctc tat aat gct tca aaa agc gga 912 PheAsp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly 290 295 300ggg aat tat gat atg agg caa ata ttt aat ggt aca gtc gtg caa aga 960 GlyAsn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg 305 310 315320 cat cca atg cat gct gtt aca ttt gtt gat aat cat gat tcg caa cct 1008His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330335 gaa gaa gct tta gag tct ttt gtt gaa gaa tgg ttc aaa cca tta gcg 1056Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345350 tat gct ttg aca tta aca cgt gaa caa ggc tac cct tct gta ttt tat 1104Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360365 gga gat tat tat ggc att cca acg cat ggt gta cca gcg atg aaa tcg 1152Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375380 aaa att gac ccg att cta gaa gcg cgt caa aag tat gca tat gga aga 1200Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg 385 390395 400 caa aat gac tac tta gac cat cat aat atc atc ggt tgg aca cgt gaa1248 Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405410 415 ggg aat aca gca cac ccc aac tcc ggt tta gct act atc atg tcc gat1296 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420425 430 ggg gca gga gga aat aag tgg atg ttt gtt ggg cgt aat aaa gct ggt1344 Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 435440 445 caa gtt tgg acc gat atc act gga aat cgt gca ggt act gtt acg att1392 Gln Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly Thr Val Thr Ile 450455 460 aat gct gat gga tgg ggt aat ttt tct gta aat gga gga tca gtt tct1440 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465470 475 480 att tgg gta aac aaa taa 1458 Ile Trp Val Asn Lys 485 <210>SEQ ID NO 24 <211> LENGTH: 485 <212> TYPE: PRT <213> ORGANISM: Bacillussp. <400> SEQUENCE: 24 His His Asn Gly Thr Asn Gly Thr Met Met Gln TyrPhe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg LeuArg Ser Asp Ala Ser 20 25 30 Asn Leu Lys Asp Lys Gly Ile Ser Ala Val TrpIle Pro Pro Ala Trp 35 40 45 Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr GlyAla Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr IleArg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Asn Gln Leu Gln Ala Ala Val AsnAla Leu Lys Ser Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met AsnHis Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Met Val Arg Ala Val GluVal Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Val Ser Gly Glu Tyr ThrIle Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn ThrHis Ser Asn Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly ValAsp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg 165 170 175 Ile Tyr Lys PheArg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp 180 185 190 Thr Glu AsnGly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205 Asp HisPro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 ThrAsn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala 245250 255 Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu260 265 270 Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His SerVal 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser LysSer Gly 290 295 300 Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr ValVal Gln Arg 305 310 315 320 His Pro Met His Ala Val Thr Phe Val Asp AsnHis Asp Ser Gln Pro 325 330 335 Glu Glu Ala Leu Glu Ser Phe Val Glu GluTrp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Thr Leu Thr Arg Glu GlnGly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro ThrHis Gly Val Pro Ala Met Lys Ser 370 375 380 Lys Ile Asp Pro Ile Leu GluAla Arg Gln Lys Tyr Ala Tyr Gly Arg 385 390 395 400 Gln Asn Asp Tyr LeuAsp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Thr AlaHis Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Ala GlyGly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 435 440 445 Gln ValTrp Thr Asp Ile Thr Gly Asn Arg Ala Gly Thr Val Thr Ile 450 455 460 AsnAla Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475480 Ile Trp Val Asn Lys 485 <210> SEQ ID NO 25 <211> LENGTH: 1458 <212>TYPE: DNA <213> ORGANISM: Bacillus sp. <220> FEATURE: <221> NAME/KEY:mat_peptide <222> LOCATION: (1)...(1458) <221> NAME/KEY: CDS <222>LOCATION: (1)...(1458) <400> SEQUENCE: 25 cac cat aat ggt acg aac ggcaca atg atg cag tac ttt gaa tgg tat 48 His His Asn Gly Thr Asn Gly ThrMet Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 cta cca aat gac gga aac cattgg aat aga tta agg tct gat gca agt 96 Leu Pro Asn Asp Gly Asn His TrpAsn Arg Leu Arg Ser Asp Ala Ser 20 25 30 aac cta aaa gat aaa ggg atc tcagcg gtt tgg att cct cct gca tgg 144 Asn Leu Lys Asp Lys Gly Ile Ser AlaVal Trp Ile Pro Pro Ala Trp 35 40 45 aag ggt gcc tct caa aat gat gtg gggtat ggt gct tat gat ctg tat 192 Lys Gly Ala Ser Gln Asn Asp Val Gly TyrGly Ala Tyr Asp Leu Tyr 50 55 60 gat tta gga gaa ttc aat caa aaa gga accatt cgt aca aaa tat gga 240 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr IleArg Thr Lys Tyr Gly 65 70 75 80 acg cgc aat cag tta caa gct gca gtt aacgcc ttg aaa agt aat gga 288 Thr Arg Asn Gln Leu Gln Ala Ala Val Asn AlaLeu Lys Ser Asn Gly 85 90 95 att caa gtg tat ggc gat gtt gta atg aat cataaa ggg gga gca gac 336 Ile Gln Val Tyr Gly Asp Val Val Met Asn His LysGly Gly Ala Asp 100 105 110 gct acc gaa atg gtt agg gcg gtt gaa gta aacccg aat aat aga aat 384 Ala Thr Glu Met Val Arg Ala Val Glu Val Asn ProAsn Asn Arg Asn 115 120 125 caa gaa gtg tcc ggt gaa tat aca att gag gcttgg aca aag ttt gac 432 Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala TrpThr Lys Phe Asp 130 135 140 ttt cct gga cga ggt aat acc cat tca aac ttcaaa tgg aga tgg tat 480 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe LysTrp Arg Trp Tyr 145 150 155 160 cac ttt gat gga gta gat tgg gat cag tcacgt aag ctg aac aat cga 528 His Phe Asp Gly Val Asp Trp Asp Gln Ser ArgLys Leu Asn Asn Arg 165 170 175 att tat aaa ttt aga ggt gat gga aaa gggtgg gat tgg gaa gtc gat 576 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly TrpAsp Trp Glu Val Asp 180 185 190 aca gaa aac ggt aac tat gat tac cta atgtat gca gat att gac atg 624 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met TyrAla Asp Ile Asp Met 195 200 205 gat cac cca gag gta gtg aat gag cta agaaat tgg ggt gtt tgg tat 672 Asp His Pro Glu Val Val Asn Glu Leu Arg AsnTrp Gly Val Trp Tyr 210 215 220 acg aat aca tta ggc ctt gat ggt ttt agaata gat gca gta aaa cat 720 Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg IleAsp Ala Val Lys His 225 230 235 240 ata aaa tac agc ttt act cgt gat tggatc aat cat gtt aga agt gca 768 Ile Lys Tyr Ser Phe Thr Arg Asp Trp IleAsn His Val Arg Ser Ala 245 250 255 act ggc aaa aat atg ttt gcg gtt gcggaa ttt tgg aaa aat gat tta 816 Thr Gly Lys Asn Met Phe Ala Val Ala GluPhe Trp Lys Asn Asp Leu 260 265 270 ggt gct att gaa aac tat tta aac aaaaca aac tgg aac cat tca gtc 864 Gly Ala Ile Glu Asn Tyr Leu Asn Lys ThrAsn Trp Asn His Ser Val 275 280 285 ttt gat gtt ccg ctg cac tat aac ctctat aat gct tca aaa agc gga 912 Phe Asp Val Pro Leu His Tyr Asn Leu TyrAsn Ala Ser Lys Ser Gly 290 295 300 ggg aat tat gat atg agg caa ata tttaat ggt aca gtc gtg caa aga 960 Gly Asn Tyr Asp Met Arg Gln Ile Phe AsnGly Thr Val Val Gln Arg 305 310 315 320 cat cca atg cat gct gtt aca tttgtt gat aat cat gat tcg caa cct 1008 His Pro Met His Ala Val Thr Phe ValAsp Asn His Asp Ser Gln Pro 325 330 335 gaa gaa gct tta gag tct ttt gttgaa gaa tgg ttc aaa cca tta gcg 1056 Glu Glu Ala Leu Glu Ser Phe Val GluGlu Trp Phe Lys Pro Leu Ala 340 345 350 tat gct ttg aca tta aca cgt gaacaa ggc tac cct tct gta ttt tat 1104 Tyr Ala Leu Thr Leu Thr Arg Glu GlnGly Tyr Pro Ser Val Phe Tyr 355 360 365 gga gat tat tat ggc att cca acgcat ggt gta cca gcg atg aaa tcg 1152 Gly Asp Tyr Tyr Gly Ile Pro Thr HisGly Val Pro Ala Met Lys Ser 370 375 380 aaa att gac ccg att cta gaa gcgcgt caa aag tat gca tat gga aga 1200 Lys Ile Asp Pro Ile Leu Glu Ala ArgGln Lys Tyr Ala Tyr Gly Arg 385 390 395 400 caa aat gac tac tta gac catcat aat atc att ggt tgg aca cgt gaa 1248 Gln Asn Asp Tyr Leu Asp His HisAsn Ile Ile Gly Trp Thr Arg Glu 405 410 415 ggg aat aca gca cac ccc aactct ggt tta gct act atc atg tcc gat 1296 Gly Asn Thr Ala His Pro Asn SerGly Leu Ala Thr Ile Met Ser Asp 420 425 430 gga gca gga gga aat aag tggatg ttt gtt ggg cgt aat aaa gct ggt 1344 Gly Ala Gly Gly Asn Lys Trp MetPhe Val Gly Arg Asn Lys Ala Gly 435 440 445 caa gtt tgg acc gat atc actgga aat cgt gca ggt act gtt acg att 1392 Gln Val Trp Thr Asp Ile Thr GlyAsn Arg Ala Gly Thr Val Thr Ile 450 455 460 aat gct gat gga tgg ggt aatttt tct gta aat gga gga tca gtt tct 1440 Asn Ala Asp Gly Trp Gly Asn PheSer Val Asn Gly Gly Ser Val Ser 465 470 475 480 att tgg gta aac aaa taa1458 Ile Trp Val Asn Lys 485 <210> SEQ ID NO 26 <211> LENGTH: 485 <212>TYPE: PRT <213> ORGANISM: Bacillus sp. <400> SEQUENCE: 26 His His AsnGly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu ProAsn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser 20 25 30 Asn LeuLys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys GlyAla Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp LeuGly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly 65 70 75 80 ThrArg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 85 90 95 IleGln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120125 Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130135 140 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu AsnAsn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp TrpGlu Val Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr AlaAsp Ile Asp Met 195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg AsnTrp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Gly Leu Asp Gly Phe ArgIle Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg AspTrp Ile Asn His Val Arg Ser Ala 245 250 255 Thr Gly Lys Asn Met Phe AlaVal Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn TyrLeu Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro LeuHis Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly 290 295 300 Gly Asn Tyr AspMet Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg 305 310 315 320 His ProMet His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 GluGlu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345 350Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370375 380 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg385 390 395 400 Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp ThrArg Glu 405 410 415 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr IleMet Ser Asp 420 425 430 Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly ArgAsn Lys Ala Gly 435 440 445 Gln Val Trp Thr Asp Ile Thr Gly Asn Arg AlaGly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser ValAsn Gly Gly Ser Val Ser 465 470 475 480 Ile Trp Val Asn Lys 485 <210>SEQ ID NO 27 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Unknown<220> FEATURE: <223> OTHER INFORMATION: degenerate primer region <223>OTHER INFORMATION: Xaa = Leu,Val,Ile <223> OTHER INFORMATION: Xaa =Ile,Leu <400> SEQUENCE: 27 Gly Ile Thr Ala Xaa Trp Xaa 1 5 <210> SEQ IDNO 28 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Unknown <220>FEATURE: <223> OTHER INFORMATION: Degenerate primer region <223> OTHERINFORMATION: Xaa = Gly,Ala <223> OTHER INFORMATION: Xaa = Val,Phe,Leu<223> OTHER INFORMATION: Xaa = Met,Leu,Ile,Phe <400> SEQUENCE: 28 ValTyr Xaa Asp Xaa Val Xaa Asn His 1 5 <210> SEQ ID NO 29 <211> LENGTH: 10<212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHERINFORMATION: Degenerated Primer region <223> OTHER INFORMATION: Xaa =Phe,Ile <223> OTHER INFORMATION: Xaa = Phe,Leu,Ile,Val <223> OTHERINFORMATION: Xaa = Ala,Val <400> SEQUENCE: 29 Asp Gly Xaa Arg Xaa AspAla Xaa Lys His 1 5 10 <210> SEQ ID NO 30 <211> LENGTH: 10 <212> TYPE:PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION:Degenerate Primer region <223> OTHER INFORMATION: Xaa = Phe,Ile <223>OTHER INFORMATION: Xaa =Phe,Leu,Ile,Val <223> OTHER INFORMATION: Xaa =Ala,Val <400> SEQUENCE: 30 Asp Gly Xaa Arg Xaa Asp Ala Xaa Lys His 1 510 <210> SEQ ID NO 31 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:Unknown <220> FEATURE: <223> OTHER INFORMATION: Degenerate Primer region<223> OTHER INFORMATION: Xaa = Asp,Glu <400> SEQUENCE: 31 Val Thr PheVal Xaa Asn His Asp 1 5 <210> SEQ ID NO 32 <211> LENGTH: 6 <212> TYPE:PRT <213> ORGANISM: Unknown <220> FEATURE: <223> OTHER INFORMATION:Degenerate Primer region <400> SEQUENCE: 32 Gly Trp Thr Arg Glu Gly 1 5<210> SEQ ID NO 33 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer188(Pst-) <400> SEQUENCE: 33 ggcgttaacc gcagcttgta ac 22 <210> SEQ ID NO34 <211> LENGTH: 51 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Primer 188cloningC <400>SEQUENCE: 34 ccgagctcgg ccggctgggc cgtcgactta tttgtttacc caaatagaaa c 51<210> SEQ ID NO 35 <211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer188cloningN <400> SEQUENCE: 35 cattctgcag cagcggcgca ccataatggt acgaacg37

What is claimed is:
 1. An isolated polypeptide having α-amylaseactivity, comprising an amino acid sequence which has at least 97%identity with amino acids 1 to 485 of SEQ ID NO:24 or SEQ ID NO:26. 2.An isolated polypeptide having α-amylase activity comprising the aminoacid sequence of SEQ ID NO: 24 or SEQ ID NO:
 26. 3. An isolatedpolypeptide having α-amylase activity consisting of the amino acidsequence of SEQ ID NO: 24 or SEQ ID NO:
 26. 4. An isolated polypeptidehaving α-amylase activity comprising an amino acid sequence which has atleast 98% identity with amino acids 1 to 485 of SEQ ID NO:24 or SEQ IDNO:26.
 5. An isolated polypeptide having α-amylase activity comprisingan amino acid sequence which has at least 99% identity with amino acids1 to 485 of SEQ ID NO:24 or SEQ ID NO:26.